Multiplex amplification detection assay ii

ABSTRACT

Provided herein is technology relating to the amplification-based detection of bisulfite-treated DNAs and particularly, but not exclusively, to methods and compositions for multiplex amplification of low-level sample DNA prior to further characterization of the sample DNA. The technology further provides methods for isolating DNA from blood or blood product samples, e.g., plasma samples.

BACKGROUND

Methods for the quantification of nucleic acids are important in manyareas of molecular biology and in particular for molecular diagnostics.At the DNA level, such methods are used, for example, to determine thepresence or absence of variant alleles, the copy numbers of genesequences amplified in a genome, and the amount, presence, or absence ofmethylation across genes or at specific loci within genes. Further,methods for the quantification of nucleic acids are used to determinemRNA quantities as a measure of gene expression.

Among the number of different analytical methods that detect andquantify nucleic acids or nucleic acid sequences, variants of thepolymerase chain reaction (PCR) have become the most powerful andwidespread technology, the principles of which are disclosed in U.S.Pat. Nos. 4,683,195; 4,683,202; and 4,965,188.

Detection of nucleic acids that are present at low levels in samples(e.g., such as DNA from a disease locus, e.g., a tumor, that iscollected from a sample that is remote from the disease locus, e.g., DNAthat finds its way into stool, sputum, urine, plasma, etc., “remote DNAsamples”) can be difficult, in part because many DNAs found in suchsamples are not only present in low amounts, they are also generallyfragmented. See, e.g., WO 2006/113770 to Ballhause, and US PatentPublication US 201110009277 A1, to Davos, each of which is incorporatedherein by reference in its entirety. For example, cell-free DNA (cfDNA)found in plasma can be highly fragmented, and much of the DNA that mightbe of interest, e.g., tumor-derived DNA can be very small, e.g., 200 orfewer nucleotides in length. Nucleic acids of this size can be lostduring routine purification, due to, e.g., poor binding to purificationcolumns or inefficient alcohol precipitation.

Analysis of such nucleic acids from such samples is especially difficultif multiple targets or loci in the nucleic acid(s) need to be detected.For example, a collected specimen having small numbers of copies of thetargets of interest often cannot be divided into a sufficient number ofaliquots to permit testing for all targets without risking the accuracyof the tests for the individual targets, e.g., by false negativeresults.

Pre-amplification of target nucleic acids (e.g., genomic DNA, cDNA,etc.) in a low-target sample may be used to enrich the DNA in the sampleprior to dividing the sample for further specific target analysis. Forexample, whole genome amplification using simple primers (e.g., randomhexamers) has been used to increase the amounts of essentially all DNAin a sample, in a manner that is not specific to any particular targetof interest. (Sigma-Aldrich's GenomePlex systems, Arneson, et al., ColdSpring Harb. Protoc.; 2008; doi:10.1101/pdb.prot4920).

Another approach is to amplify one or more regions of particularinterest in a semi-targeted manner, to produce a mixture of amplifiedfragments (amplicons) that contains the different mutations or loci thatwill be further analyzed. Successive rounds of amplification using thesame primers are prone to high background of non-specific amplification,and the production of artifacts, e.g., artificially recombinedmolecules, high non-specific background, and biased amplification ofdifferent intended targets. Thus, such pre-amplification PCR istypically carried out under special conditions e.g., a limited number ofcycles, and/or using a low concentration of primers (e.g., 10 to 20-foldlower than in standard PCR) to avoid increases in non-specificbackground amplification, as use of concentrations over about 160 nM ofeach primer in multiplex pre-amplification has been shown to increaseamplification background in negative control reactions (see, e.g.,Andersson, et al., Expert Rev. Mol. Diagn. Early online, 1-16 (2015)).

After a first round of amplification in a multiplex PCR, pre-amplifiedDNA is typically diluted and aliquoted into new amplification reactionsfor quantitative or qualitative PCR analysis using conditions typical ofstandard PCR, e.g., higher concentrations of reagents and larger numbersof cycles, and the second amplification is generally carried out usingdifferent primer pairs, e.g., “nested” primers that anneal to siteswithin the pre-amplified fragments, rather than annealing to theoriginal primer sites at the ends of the amplicons.

When DNA is to be examined for methylation, the analysis is furthercomplicated by the fact that commonly used processes for preparingsamples for methylation detection typically result in substantial lossesof sample DNA. For example, bisulfite treatment is typically used toconvert unmethylated cytosine residues to uracil residues, but theprocess typically results in only about 30% recovery of the input DNA.In addition, amplification of DNA after treatment with bisulfite isespecially challenging. For example, the conversion of unmethylatedcytosines reduces the complexity of the DNA sequences and the treatmentitself is known to cause significant damage to the DNA, e.g., strandbreakage, both of which can contribute to increased background inamplification reactions, especially in multiplexed amplifications.

SUMMARY

In the course of development of methods described herein, it has beendetermined that bisulfite-treated DNA from low-target samples can bepre-amplified and amplified for real-time detection without the need forwhole-genome pre-amplification and without the use of nested orsemi-nested primers. Surprisingly, the targeted pre-amplification can bemultiplexed using a combination of the same primer pairs that will beused in a second round of amplification of individual target loci, e.g.,in a quantitative allele-specific real-time target and signalamplification (QuARTS) assay (see, e.g., U.S. Pat. Nos. 8,361,720,8,715,937 and 8,916,344), which combines PCR target amplification andFEN-1-mediated flap cleavage for signal amplification. In anyembodiment, the method may employ a flap oligonucleotide that has atarget-specific region of at least 13 bases in length, e.g., 13 to 30,14 to 30, or 15 to 30 bases in length.

In some embodiments, the technology provides a method of analyzingsamples such that a plurality of different targets that are present inlow copy number may be individually detected with reduced risk of falsenegative results due to sample splitting. For example, in someembodiments, the technology provides a method of analyzing a sample formultiple target nucleic acids, comprising:

-   -   a) providing a sample having volume x, the sample comprising        bisulfite-treated DNA suspected of containing one or more of a        plurality of n different target regions, wherein at least one of        said target regions is a low-copy target that, if present in        said sample, is present in said sample at a copy number such        that:        -   i) among n fractions of said sample each having a volume of            x/n, said low copy target is absent from one or more of said            n fractions, or        -   ii) among n fractions of said sample each having a volume of            x/n, said low copy target in one or more of said n fractions            is below a level of sensitivity of a detection assay for            said low copy target;    -   b) treating said volume x of said sample to an amplification        reaction under conditions wherein said n different target        regions, if present in said sample, are amplified to form a        pre-amplified mixture having volume y;    -   c) partitioning said pre-amplified mixture into a plurality of        different detection assay reaction mixtures, wherein each        detection assay reaction mixture comprises a portion of said        pre-amplified mixture that has a volume of y/n or less, and        wherein said low-copy target, if present in said sample at step        a), is present in each of said detection assay reaction        mixtures; and    -   d) conducting a plurality of detection assays with said        detection assay reaction mixtures, wherein said different target        regions, if present in said sample at step a), are detected in        said detection assay reaction mixtures, wherein the detection        assays are PCR-flap assays that employ flap oligonucleotides        that have a target-specific region of at least 13 bases in        length.

In some embodiments, the bisulfite treated DNA is from a human subject.In certain preferred embodiments, the sample is prepared from a bodyfluid of a subject, preferably a body fluid comprising plasma. In someembodiments, the bisulfite treated DNA is circulating cell-free DNA(cfDNA) isolated from plasma, e.g., cell-free DNA of less than 200 basepairs in length. In particularly preferred embodiments, cell-free DNA isisolated from plasma by a method comprising combining the plasma samplewith a protease (e.g., Pronase, proteinase K) and a first lysis reagentthat comprises guanidine thiocyanate and non-ionic detergent to form amixture in which proteins are digested by the protease, then addingsilica particles and a second lysis reagent, with the second lysisreagent comprising a mixture of guanidine thiocyanate, non-ionicdetergent, and isopropyl alcohol, under conditions in which DNA is boundto the silica particles. In certain embodiments, the non-ionicdetergents in the first lysis reagent and the second lysis reagent arethe same or different, and are selected from, e.g., polyethylene glycolsorbitan monolaurate (Tween-20), octylphenoxypolyethoxyethanol (NonidetP-40), and octylphenoxy poly(ethyleneoxy) ethanol, branched (IGEPALCA-630).

The method further comprises separating the silica particle with boundDNA from the mixture, washing the separated silica particles with boundDNA with a first wash solution comprising guanidine hydrochloride orguanidine thiocyanate and ethyl alcohol, separating the silica particleswith bound DNA from the first wash solution and washing the silicaparticles with bound DNA with a second wash solution comprising abuffer, e.g., Tris pH 8.0 and ethyl alcohol. In preferred embodiments,the silica particles with bound DNA are washed multiple times, e.g., 2to 6 times, with the second wash buffer. In particularly preferredembodiments, each wash uses a smaller volume of the second wash bufferthan the prior wash with that buffer. In some embodiments the washedsilica particles are separated from the last wash buffer treatment andthe DNA is eluted from the silica particles, e.g., with an elutionbuffer, such as 10 mM Tris-HCl pH 8.0, 0.1 mM EDTA. In preferredembodiments, the silica particles with bound DNA are dried, e.g., byheating to about 70° C., prior to elution of the DNA.

The technology is not limited to any particular sample size, but itfinds particular application in samples in which low-copy targets arepresent in large samples. For example, in some embodiments, thebisulfite treated DNA is prepared from a body fluid, e.g., a plasmasample, having a starting volume of at least one mL, preferably at least5 mL, more preferably at least 10 mL, and/or wherein said volume x ofthe sample of bisulfite treated DNA is at least 10 μl, preferably atleast 25 μl, more preferably at least 50 μl, more preferably at least100 μl. In preferred embodiments, the volume of treated DNA sample thatis present in the pre-amplification reaction is at least 5%, preferablyat least 10%-60%, preferably 15%-55%, more preferably about 20%-50% ofthe total volume of the amplification reaction.

The invention is not limited to a particular number of fractions intowhich the sample is divided. In some embodiments, n (the number offractions) is at least 3, preferably at least 5, more preferably atleast 10, more preferably at least 20, more preferably at least 100.

In some embodiments, the technology provides a method for analyzingmultiple target nucleic acids in a sample using a PCR pre-amplificationand a PCR-flap assay, the method comprising:

-   -   a) providing bisulfite-treated DNA (in preferred embodiments,        comprising human DNA) comprising a plurality of different target        regions in a first reaction mixture comprising PCR amplification        reagents, wherein said PCR amplification reagents comprise:        -   i) a plurality of different primer pairs for amplifying said            plurality of different target regions, if present in said            sample, from said bisulfite-treated DNA;        -   ii) thermostable DNA polymerase;        -   iii) dNTPs; and        -   iv) a buffer comprising Mg⁺⁺    -   b) exposing said first reaction mixture to thermal cycling        conditions wherein a plurality of different target regions, if        present in the sample, are amplified to produce a pre-amplified        mixture, and wherein said thermal cycling conditions are limited        to a number of thermal cycles that maintain amplification in an        exponential range, preferably fewer than 20, more preferably        fewer than 15, more preferably 10 or fewer thermal cycles;    -   c) partitioning said pre-amplified mixture into a plurality of        PCR-flap assay reaction mixtures, wherein each PCR-flap assay        reaction mixture comprises:        -   i) an additional amount of a primer pair selected from said            plurality of different primer pairs of step a) i);        -   ii) thermostable DNA polymerase;        -   iii) dNTPs;        -   iv) said buffer comprising Mg⁺⁺        -   v) a flap endonuclease, preferably a FEN-1 endonuclease;        -   vi) a flap oligonucleotide that has a target-specific region            of at least 13 bases in length, and        -   vi) a hairpin oligonucleotide comprising a region that is            complimentary to a portion of said flap oligonucleotide,            preferably a FRET cassette oligonucleotide; and    -   d) detecting amplification of one or more different target        regions from said bisulfite-treated DNA during PCR-flap assay        reactions by detecting cleavage of said hairpin oligonucleotide        by said flap endonuclease.

In preferred embodiments, the FEN-1 endonuclease is a thermostableFEN-1, preferably from an archaeal organism, e.g., Afu FEN-1.

In some embodiments, the pre-amplified mixtures described above arediluted with a diluent prior to partitioning into PCR-flap assayreaction mixtures, while in some embodiments, the pre-amplified mixtureis added directly to a PCR-flap assay reaction mixture without priordilution.

In some embodiments, essentially the primers used in the PCR-flap assayreaction are used at the same concentrations at which those particularprimers were used in the first reaction mixture, excluding any primerscarried over from the first reaction. For example, in some embodiments,the primers in the additional amount of a primer pair added to thePCR-flap assay reaction mixture are added to a concentration such thatthe concentration of the added primers in the PCR-flap assay (i.e., notcounting primers coming from the pre-amplified mixture) is essentiallythe same as the concentration of the primers of that primer pair in saidPCR amplification reagents. In other embodiments, the primers in theadditional amount of a primer pair added to the PCR-flap assay reactionmixture are added to a concentration such that the concentration of theadded primers in the PCR-flap assay are at a lower or a higherconcentration than the concentration of the primers of that primer pairin the first reaction mixture.

While the method is not limited to a particular concentration of Mg⁺⁺ inthe buffer used in said first reaction mixture and in the PCR-flap assayreaction mixture, in preferred embodiments, the buffer comprises atleast 3 mM Mg⁺⁺, preferably greater than 4 mM Mg⁺⁺, more preferablygreater than 5 mM Mg⁺⁺, more preferably greater than 6 mM Mg⁺⁺, morepreferably between approximately 7 mM and 7.5 mM Mg^(++.) In certainembodiments, the buffer contains less than about 1 mM KCl. In preferredembodiments, the buffer comprises 10 mM 3-(n-morpholino) propanesulfonicacid (MOPS) buffer and 7.5 mM MgCl₂.

In some embodiments, the first reaction mixture and/or said plurality ofPCR-flap assay reaction mixtures comprise exogenous non-target DNA,preferably bulk fish DNA.

In some embodiments, the thermostable DNA polymerase is a eubacterialDNA polymerase, preferably from genus Thermus, more preferably fromThermus aquaticus. In some embodiments, the DNA polymerase is modifiedfor hot start PCR, e.g., though the use of a reagent, e.g., an antibody,chemical adduct, etc., such that the DNA polymerase is activated uponheating.

In certain embodiments, the bisulfite-treated DNA comprises human DNA,and the plurality of different target regions comprises target regionsselected from the group consisting of SFMBT2, VAV3, BMP3, and NDRG4. Insome embodiments, a plurality of different primer pairs are directed toat least two, preferably at least three, more preferably all four ofthese target regions.

In some embodiments, the plurality of different target regions comprisea reference target region, and in certain preferred embodiments, thereference target region comprises β-actin and and/or ZDHHCl, and/orB3GALT6.

In some embodiments, at least one of the plurality of different primerpairs is selected to produce an amplicon from a target region that isless than about 100 base pairs long, preferably less than about 85 basepairs long. In certain preferred embodiments, all of the differentprimer pairs are selected to produce an amplicon from a target regionthat is less than about 100 base pairs long.

In some embodiments, methods provided herein are directed to amplifyingsubstantially all of the bisulfite-treated DNA produced during theprocess of a sample, e.g., a sample of bodily fluid. In someembodiments, the preparation of bisulfite treated DNA constitutes asubstantial fraction of the first reaction mixtures, e.g., in someembodiments, the volume of the sample comprising bisulfite-treated DNAin the first reaction mixture constitutes at least 20-50% of the totalvolume of the first reaction mixture. For example, in some embodiments,the volume of bisulfite-treated DNA in the first reaction mixture is atleast 5%, preferably at least 10%-60%, preferably 15%-55%, morepreferably between about 20%-50% of the total volume of the firstreaction mixture.

In some embodiments, methods of the technology provide a method foranalyzing multiple target nucleic acids in a sample of human plasmausing a PCR pre-amplification and a PCR-flap assay, the methodcomprising:

-   -   a) providing bisulfite-treated DNA prepared from at least 1 mL        of plasma, the bisulfite treated DNA comprising a plurality of        different target regions in a first reaction mixture comprising        PCR amplification reagents, wherein said PCR amplification        reagents comprise:        -   i) a plurality of different primer pairs for amplifying said            plurality of different target regions, said target regions            selected from SFMBT2, VAV3, BMP3, and NDRG4, if present in            said sample, from said bisulfite-treated DNA, wherein each            of said plurality of different primer pairs is selected to            produce an amplicon from a target region that is less than            about 100 base pairs long;        -   ii) DNA polymerase from Thermus aquaticus;        -   iii) dNTPs; and        -   iv) a buffer comprising 7.5 mM Mg⁺⁺    -   b) exposing said first reaction mixture to thermal cycling        conditions wherein a plurality of different target regions, if        present in the sample, are amplified to produce a pre-amplified        mixture, and wherein said thermal cycling conditions are limited        to a number of thermal cycles that maintain amplification in an        exponential range, preferably fewer than 20, more preferably        fewer than 15, more preferably 10 or fewer thermal cycles;    -   c) partitioning said pre-amplified mixture into a plurality of        PCR-flap assay reaction mixtures, wherein each PCR-flap assay        reaction mixture comprises:        -   i) an additional amount of a primer pair selected from said            plurality of different primer pairs of step a) i);        -   ii) DNA polymerase from Thermus aquaticus;        -   iii) dNTPs;        -   iv) said buffer comprising 7.5 mM Mg⁺⁺        -   v) a thermostable FEN-1 flap endonuclease;        -   vi) a flap oligonucleotide that has a target-specific region            of at least 13 bases in length, and        -   vi) a FRET cassette oligonucleotide comprising a region that            is complimentary to a portion of said flap oligonucleotide;            and    -   d) detecting amplification of one or more the different target        regions selected from SFMBT2, VAV3, BMP3, and NDRG4 during        PCR-flap assay reactions.

In preferred embodiments, the plurality of different target regionscomprise a reference target region, preferably comprising comprisesβ-actin and/or ZDHHCl. In certain embodiments, one or more of the targetregions and/or primers pairs is selected from the target regions andprimer pairs depicted in FIGS. 5A-5F.

Also provided herein are improved methods for isolating DNA, e.g.,cell-free DNA from blood or blood fractions, e.g., plasma or serum. Forexample, embodiments provide methods of processing a plasma sample, themethod comprising combining the plasma sample with a protease and afirst lysis reagent that comprises guanidine thiocyanate and non-ionicdetergent to form a mixture in which proteins are digested by theprotease, then adding mixable silica particles and a second lysisreagent, with the second lysis reagent comprising a mixture of guanidinethiocyanate, non-ionic detergent, and isopropyl alcohol, underconditions in which DNA is bound to the silica particles. In certainembodiments, the non-ionic detergents in the first lysis reagent and thesecond lysis reagent are the same or different, and are selected from,e.g., polyethylene glycol sorbitan monolaurate (Tween-20),octylphenoxypolyethoxyethanol (Nonidet P-40), and octylphenoxypoly(ethyleneoxy) ethanol, branched (IGEPAL CA-630). In certainpreferred embodiments, the silica particles are magnetic particles.

The method further comprises separating the silica particles with boundDNA from the mixture, washing the separated silica particles with boundDNA with a first wash solution comprising guanidine hydrochloride orguanidine thiocyanate and ethyl alcohol, separating the silica particleswith bound DNA from the first wash solution and washing the silicaparticles with bound DNA with a second wash solution comprising abuffer, e.g., Tris pH 8.0, and ethyl alcohol. In preferred embodiments,the silica particles with bound DNA are washed multiple times, e.g., 2to 6 times, with the second wash buffer. In particularly preferredembodiments, each wash uses a smaller volume of the second wash bufferthan the prior wash with that buffer. In some embodiments the washedsilica particles are separated from the last wash buffer treatment andthe DNA is eluted from the silica particles, e.g., with water or with anelution buffer, such as 10 mM Tris-HCl pH 8.0, 0.1 mM EDTA. In preferredembodiments, the silica particles with bound DNA are dried after thelast wash step, e.g., by heating (to, for example, 37° C. to 75° C.,preferably about 45° C. to 70° C., more preferably about 70° C.), priorto elution of the DNA.

During development of the technology it was discovered that use twodifferent lysis reagents, added at different times in the procedure,improves yield of DNA from plasma. In preferred embodiments, an aliquotof the second lysis reagent is added after the mixture comprising thefirst lysis reagent and protease have incubated, e.g., for about 5 to 60minutes, preferably 30 to 60 minutes, at room temperature to 55° C. Inpreferred embodiments, the mixture is incubated at room temperature. Incertain embodiments, the first lysis reagent comprises guanidinethiocyanate and a non-ionic detergent, and the second lysis reagentcomprises guanidine thiocyanate, a non-ionic detergent, and an alcohol.In preferred embodiments, the first lysis reagent comprises about 4.3 Mguanidine thiocyanate and 10% w:v IGEPAL CA-630, and in someembodiments, the second lysis reagent comprises 4.3 M guanidinethiocyanate and 10% w:v IGEPAL CA-630 combined with isopropyl alcohol.

During development of the technology it was discovered that use twodifferent wash solutions at different steps in the procedure improvedyield of DNA from plasma. In some embodiments, a first wash solution,used as described above, comprises guanidine hydrochloride or guanidinethiocyanate and ethyl alcohol, and the second wash solution comprises abuffer and ethyl alcohol. In particularly preferred embodiments, thefirst wash solution comprises about 3 M guanidine hydrochloride orguanidine thiocyanate and about 57% ethyl alcohol and the second washsolution, used as described above, comprises about 80% ethyl alcohol andabout 20% 10 mM Tris pH 8.0 buffer.

In particularly preferred embodiments, all lysis steps and wash stepsare conducted at room temperature.

In some embodiments, the plasma sample is mixed with a DNA processcontrol, e.g., a DNA that does not cross-react with assays configured todetect DNA found in the plasma sample. For example, in some embodimentsthe plasma is human plasma and the DNA process control comprises azebrafish RASSF1 sequence. In preferred embodiments, the DNA processcontrol is synthetic DNA, e.g., a synthetic DNA fragment comprising azebrafish RASSF1 sequence. In particularly preferred embodiments, theDNA process control is double stranded. In preferred embodiments, theprocess control is added to the plasma sample prior to extraction of DNAfrom the sample, e.g., along with the first or second lysis reagentadditions.

In some embodiments, bulk exogenous DNA, e.g., DNA that does notcross-react with assays configured to detect DNA found in the plasmasample, is added to the plasma sample. For example, in preferredembodiments, the plasma is human plasma and bulk fish DNA, e.g., genomicDNA from salmon, is added to the sample.

Embodiments of the methods provided herein find particular use in theprocessing of relatively large plasma samples, e.g., greater than 1 mL.In preferred embodiments, the plasma sample has a volume of at least 2mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, or at least 10 mL, or anyfractional volume therebetween. In some embodiments the plasma sample isgreater than 10 mL in volume.

In some embodiments, the method further comprises analyzing the isolatedDNA sample for particular target nucleic acids. In preferredembodiments, the method comprises analyzing the isolated DNA for aplurality of methylated target nucleic acids, the method comprisingtreating the isolated DNA sample with bisulfite to produce abisulfite-treated DNA sample, treating the bisulfite-treated DNA sampleto an amplification reaction under conditions wherein a plurality ofdifferent target regions (e.g., 2, 3, 4, 5, etc. target regions), ifpresent in the sample, are amplified to form an amplified mixture.

In certain preferred embodiments the method further comprisespartitioning the amplified mixture into a plurality of differentdetection assay reaction mixtures and conducting a plurality ofdifferent detection assays with the detection assay reaction mixtures,wherein the plurality of different target regions, if present in thesample, are detected in one or more of the plurality of differentdetection assay reaction mixtures. In preferred embodiments, theplurality of different target regions comprises at least 5 differenttarget regions.

Provided herein are kits and systems for performing methods describedherein. In some embodiments the technology provides a kit for isolatingDNA from plasma, the kit comprising, e.g.:

-   -   a) a first lysis reagent comprising guanidine thiocyanate and a        non-ionic detergent or components for preparing the first lysis        reagent;    -   b) a second lysis reagent comprising guanidine thiocyanate, a        non-ionic detergent, and isopropanol, or components for        preparing the second lysis reagent;    -   c) a first wash solution comprising guanidine hydrochloride or        guanidine thiocyanate and ethyl alcohol, or components for        preparing the first wash solution;    -   d) a second wash solution comprising Tris buffer and ethyl        alcohol, or components for preparing the second wash solution;        and    -   e) silica particles.

In preferred embodiments, the non-ionic detergent comprises IGEPALCA-630. In some embodiments, the silica particles are magneticparticles, and in particularly preferred embodiments, the kit comprisesa magnet, e.g., for separating the particles during steps of theprocedure. In some embodiments, the kit further comprises an elutionbuffer or components for preparing the elution buffer.

In some embodiments the kit further comprises a DNA process control,e.g., a DNA process control comprising a zebrafish RASSF1 sequence. Insome embodiments the kit further comprises a preparation of bulk fishDNA, and in particularly preferred embodiments, the DNA process controlis in a preparation of bulk fish DNA.

In some embodiments the technology provides a system for processing aplasma sample, the system comprising:

-   -   a) a first lysis reagent comprising guanidine thiocyanate and a        non-ionic detergent or components for preparing the first lysis        reagent;    -   b) a second lysis reagent comprising guanidine thiocyanate, a        non-ionic detergent, and isopropanol, or components for        preparing the second lysis reagent;    -   c) a first wash solution comprising guanidine hydrochloride or        guanidine thiocyanate and ethyl alcohol, or components for        preparing the first wash solution;    -   d) a second wash solution comprising Tris buffer and ethyl        alcohol, or components for preparing the second wash solution;        and    -   e) silica particles.

In preferred embodiments, the non-ionic detergent comprises IGEPALCA-630.

In some embodiments the system further comprises an elution buffer orcomponents for preparing said elution buffer.

In some embodiments the system further comprises a DNA process control,e.g., a DNA process control comprising a zebrafish RASSF1 sequence. Insome embodiments the system further comprises a preparation of bulk fishDNA, and in particularly preferred embodiments, the DNA process controlis in a preparation of bulk fish DNA.

In some embodiments, the system further comprises one or more of: amagnet, a vessel for processing plasma, and/or a vessel or plate forreceiving purified DNA. In some embodiments, the system comprises adevice for performing all or part of the steps, e.g., a device such as aSTARlet automated platform.

In some embodiments, the system further comprises reagents for analysisof DNA isolated from plasma. For example, in certain embodiments, thesystem comprises reagents for treating DNA with bisulfite to producebisulfite-treated DNA, e.g., a bisulfite reagent, a desulfonationreagent, and materials for purifying bisulfite-treated DNA (e.g., silicabeads, a binding buffer, a solution comprising bovine serum albuminand/or casein, e.g., as described in U.S. Pat. No. 9,315,853,incorporated herein by reference).

In preferred embodiments, the system further comprises DNA analysisreagents, e.g., PCR amplification reagents and/or flap assay reagents.In particularly preferred embodiments, the system comprises PCRamplification reagents comprising:

-   -   i) a plurality of different primer pairs for amplifying a        plurality of different target regions, if present in said        plasma;    -   ii) thermostable DNA polymerase;    -   iii) dNTPs; and    -   iv) a buffer comprising Mg⁺⁺

In some embodiments, the system further comprises PCR-flap assayreagents. In certain preferred embodiments, the PCR flap assay reagentscomprise:

-   -   i) a plurality of different primer pairs for amplifying a        plurality of different target regions, if present in said        plasma;    -   ii) thermostable DNA polymerase;    -   iii) dNTPs;    -   iv) a buffer comprising Mg⁺⁺;    -   v) a flap endonuclease;    -   vi) a flap oligonucleotide that has a target-specific region of        at least 13 bases in length, and    -   vi) a hairpin oligonucleotide comprising a region that is        complimentary to a portion of said flap oligonucleotide.

In still further embodiments, the system comprises a thermal cycler forconducting PCR amplification and/or PCR flap assay reactions. Inpreferred embodiments, the thermal cycler is configured to detectsignal, e.g., fluorescence, during the course of amplification reactionsconducted with the assay reagents.

Additional embodiments will be apparent to persons skilled in therelevant art based on the teachings contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presenttechnology will become better understood with regard to the followingdrawings:

FIG. 1 provides a schematic diagram of a combined PCR-invasive cleavageassay (“PCR-flap assay”), e.g., a QuARTS assay.

FIG. 2 provides a schematic diagram of nested PCR combined with aPCR-flap assay, showing a first amplification (or pre-amplification)using outer primers, followed by a PCR-flap assay using a second pair ofprimers having binding sites within the sites of the outer primers. Thesmaller amplicon produced in the second amplification is shown at thebottom. The FRET-cassette portion of the reaction is not shown.

FIG. 3 provides a schematic diagram of a PCR pre-amplification followedby a PCR-flap assay in which the pre-amplification and the PCR-flapassay use the same primer pair, and producing copies of the sameamplicon. The FRET-cassette portion of the reaction is not shown.

FIG. 4 provides a schematic diagram of a multiplex pre-amplification inwhich a plurality of different target regions in a sample are amplifiedin a single multiplexed PCR reaction containing primer pairs for each ofthe different target regions, followed by individual PCR-flap assayreactions in which each PCR flap assay uses only the primer pairspecific for the target locus to be detected in the final PCR-flap assayreaction.

FIGS. 5A-5F show nucleic acid sequences for analysis of methylationusing the combination of bisulfite conversion, pre-amplification, andPCR-flap assay detection. Each panel shows one strand of the DNA targetregion prior to bisulfite treatment and the expected sequence of thatregion upon conversion with bisulfite reagent, with convertedunmethylated C residues shown as ‘T’s. The primer binding sites forouter primers and for PCR-flap assay inner primers (as would be used fora nested assay design) are shown boxed. Each figure also shows analignment of the PCR-flap assay primers and flap probe on a segment ofthe converted sequence. FIGS. 5A-5F show target regions of markersSFMBT2, VAV3, BMP3, NDRG4, and reference DNAs β-actin, and ZDHHCl,respectively. The ‘arms’ on the flap oligonucleotides used in thePCR-flap assay are as follows: Arm 1 is 5′-CGCCGAGG-3′; Arm 3 is5′-GACGCGGAG-3′; and Arm 5 is 5′-CCACGGACG-3′.

FIG. 6 shows a table comparing detection of the indicatedbisulfite-treated target DNAs pre-amplified using outer primers fordifferent numbers of cycles, followed by PCR-flap assay amplificationand detection using nested (inner) primers. Comparative assays used aQuARTS PCR-flap assay directly on the bisulfite-treated DNA, withoutpre-amplification.

FIG. 7 compares results using nested or non-nested amplification primerconfigurations as shown in FIGS. 5A-5F, and compares different primerconcentrations and different buffers in the PCR pre-amplification step,as described in Example 3.

FIGS. 8A-8C show the results of using different numbers of cycles in thepre-amplification phase of the assay. FIG. 8A shows the number ofstrands expected for each of the target types in normal plasma samplesor in plasma samples spiked with known amounts of target DNAs, witheither no pre-amplification, or with 5, 10, 20 or 25 cycles ofamplification. FIG. 8B compares the number of strands detection in eachreaction under the conditions show, as described in Example 4.

FIG. 9 shows the results of using a non-nested multiplexpre-amplification on DNA isolated from stool, as described in Example 5.

FIGS. 10A-10I show the results of using a non-nested multiplexpre-amplification on DNA isolated from plasma, as described in Example6.

FIGS. 11A-11C show graphs comparing different plasma isolationconditions on the yield of β-actin DNA (untreated and bisulfiteconverted after extraction) and the B3GALT6 gene (bisulfite convertedafter extraction, as described in Example 8.

FIGS. 12A-12C show graphs comparing different plasma isolationconditions on the yield of β-actin DNA (untreated and bisulfiteconverted after extraction) and the B3GALT6 gene (bisulfite convertedafter extraction, as described in Example 10.

FIGS. 13A-13F show a table of nucleic acid sequences relating toembodiments herein.

FIG. 14 shows representative results obtained from Experiment 1 ofExample 12.

FIG. 15 shows representative results obtained from Experiment 2 ofExample 12.

FIG. 16 shows representative results obtained from Experiment 1 ofExample 13.

FIG. 17 shows representative results obtained from Experiment 2 ofExample 13.

It is to be understood that the figures are not necessarily drawn toscale, nor are the objects in the figures necessarily drawn to scale inrelationship to one another. The figures are depictions that areintended to bring clarity and understanding to various embodiments ofapparatuses, systems, and methods disclosed herein. Wherever possible,the same reference numbers will be used throughout the drawings to referto the same or like parts. Moreover, it should be appreciated that thedrawings are not intended to limit the scope of the present teachings inany way.

Definitions

To facilitate an understanding of the present technology, a number ofterms and phrases are defined below. Additional definitions are setforth throughout the detailed description.

Throughout the specification and claims, the following terms take themeanings explicitly associated herein, unless the context clearlydictates otherwise. The phrase “in one embodiment” as used herein doesnot necessarily refer to the same embodiment, though it may.Furthermore, the phrase “in another embodiment” as used herein does notnecessarily refer to a different embodiment, although it may. Thus, asdescribed below, various embodiments of the technology may be readilycombined, without departing from the scope or spirit of the technology.

In addition, as used herein, the term “or” is an inclusive “or” operatorand is equivalent to the term “and/or” unless the context clearlydictates otherwise. The term “based on” is not exclusive and allows forbeing based on additional factors not described, unless the contextclearly dictates otherwise. In addition, throughout the specification,the meaning of “a”, “an”, and “the” include plural references. Themeaning of “in” includes “in” and “on.”

The transitional phrase “consisting essentially of” as used in claims inthe present application limits the scope of a claim to the specifiedmaterials or steps “and those that do not materially affect the basicand novel characteristic(s)” of the claimed invention, as discussed inIn re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976). Forexample, a composition “consisting essentially of” recited elements maycontain an unrecited contaminant at a level such that, though present,the contaminant does not alter the function of the recited compositionas compared to a pure composition, i.e., a composition “consisting of”the recited components.

As used herein in reference to non-target DNA, the term “exogenous”refers to non-target DNA that is isolated and purified from a sourceother than the source or sample containing the target DNA. For example,purified fish DNA is exogenous DNA with respect to a sample comprisinghuman target DNA, e.g., as described in U.S. Pat. No. 9,212,392, whichis incorporated herein by reference. Exogenous DNA need not be from adifferent organism than the target DNA. For example, purified fish DNAobtained commercially would be exogenous if added to a reactionconfigured to detect a target nucleic acid in a sample from a particularfish. In preferred embodiments, exogenous DNA is selected to beundetected by an assay configured to detect and/or quantify the targetnucleic acid in the reaction in to which the exogenous DNA is added.

As used herein, a “DNA fragment” or “small DNA” or “short DNA” means aDNA that consists of no more than approximately 200 base pairs ornucleotides in length.

The term “primer” refers to an oligonucleotide that is capable of actingas a point of initiation of synthesis when placed under conditions inwhich primer extension is initiated. An oligonucleotide “primer” mayoccur naturally, as in a purified restriction digest or may be producedsynthetically. In some embodiments, an oligonucleotide primer is usedwith a template nucleic acid and extension of the primer is templatedependent, such that a complement of the template is formed.

The term “amplifying” or “amplification” in the context of nucleic acidsrefers to the production of multiple copies of a polynucleotide, or aportion of the polynucleotide, typically starting from a small amount ofthe polynucleotide (e.g., a single polynucleotide molecule), where theamplification products or amplicons are generally detectable.Amplification of polynucleotides encompasses a variety of chemical andenzymatic processes. The generation of multiple DNA copies from one or afew copies of a target or template DNA molecule during a polymerasechain reaction (PCR) or a ligase chain reaction (LCR; see, e.g., U.S.Pat. No. 5,494,810; herein incorporated by reference in its entirety)are forms of amplification. Additional types of amplification include,but are not limited to, allele-specific PCR (see, e.g., U.S. Pat. No.5,639,611; herein incorporated by reference in its entirety), assemblyPCR (see, e.g., U.S. Pat. No. 5,965,408; herein incorporated byreference in its entirety), helicase-dependent amplification (see, e.g.,U.S. Pat. No. 7,662,594; herein incorporated by reference in itsentirety), hot-start PCR (see, e.g., U.S. Pat. Nos. 5,773,258 and5,338,671; each herein incorporated by reference in their entireties),intersequence-specific PCR, inverse PCR (see, e.g., Triglia, et al.,(1988) Nucleic Acids Res., 16:8186; herein incorporated by reference inits entirety), ligation-mediated PCR (see, e.g., Guilfoyle, R. et al.,Nucleic Acids Research, 25:1854-1858 (1997); U.S. Pat. No. 5,508,169;each of which are herein incorporated by reference in their entireties),methylation-specific PCR (see, e.g., Herman, et al., (1996) PNAS 93(13)9821-9826; herein incorporated by reference in its entirety), miniprimerPCR, multiplex ligation-dependent probe amplification (see, e.g.,Schouten, et al., (2002) Nucleic Acids Research 30(12): e57; hereinincorporated by reference in its entirety), multiplex PCR (see, e.g.,Chamberlain, et al., (1988) Nucleic Acids Research 16(23) 11141-11156;Ballabio, et al., (1990) Human Genetics 84(6) 571-573; Hayden, et al.,(2008) BMC Genetics 9:80; each of which are herein incorporated byreference in their entireties), nested PCR, overlap-extension PCR (see,e.g., Higuchi, et al., (1988) Nucleic Acids Research 16(15) 7351-7367;herein incorporated by reference in its entirety), real time PCR (see,e.g., Higuchi, et al., (1992) Biotechnology 10:413-417; Higuchi, et al.,(1993) Biotechnology 11:1026-1030; each of which are herein incorporatedby reference in their entireties), reverse transcription PCR (see, e.g.,Bustin, S. A. (2000) J. Molecular Endocrinology 25:169-193; hereinincorporated by reference in its entirety), solid phase PCR, thermalasymmetric interlaced PCR, and Touchdown PCR (see, e.g., Don, et al.,Nucleic Acids Research (1991) 19(14) 4008; Roux, K. (1994) Biotechniques16(5) 812-814; Hecker, et al., (1996) Biotechniques 20(3) 478-485; eachof which are herein incorporated by reference in their entireties).Polynucleotide amplification also can be accomplished using digital PCR(see, e.g., Kalinina, et al., Nucleic Acids Research. 25; 1999-2004,(1997); Vogelstein and Kinzler, Proc Natl Acad Sci USA. 96; 9236-41,(1999); International Patent Publication No. WO05023091A2; US PatentApplication Publication No. 20070202525; each of which are incorporatedherein by reference in their entireties).

The term “polymerase chain reaction” (“PCR”) refers to the method of K.B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, thatdescribe a method for increasing the concentration of a segment of atarget sequence in a mixture of genomic or other DNA or RNA, withoutcloning or purification. This process for amplifying the target sequenceconsists of introducing a large excess of two oligonucleotide primers tothe DNA mixture containing the desired target sequence, followed by aprecise sequence of thermal cycling in the presence of a DNA polymerase.The two primers are complementary to their respective strands of thedouble stranded target sequence. To effect amplification, the mixture isdenatured and the primers then annealed to their complementary sequenceswithin the target molecule. Following annealing, the primers areextended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing, and polymeraseextension can be repeated many times (i.e., denaturation, annealing andextension constitute one “cycle”; there can be numerous “cycles”) toobtain a high concentration of an amplified segment of the desiredtarget sequence. The length of the amplified segment of the desiredtarget sequence is determined by the relative positions of the primerswith respect to each other, and therefore, this length is a controllableparameter. By virtue of the repeating aspect of the process, the methodis referred to as the “polymerase chain reaction” (“PCR”). Because thedesired amplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified” and are “PCR products” or “amplicons.” Those of skillin the art will understand the term “PCR” encompasses many variants ofthe originally described method using, e.g., real time PCR, nested PCR,reverse transcription PCR (RT-PCR), single primer and arbitrarily primedPCR, etc.

As used herein, the term “nucleic acid detection assay” refers to anymethod of determining the nucleotide composition of a nucleic acid ofinterest. Nucleic acid detection assay include but are not limited to,DNA sequencing methods, probe hybridization methods, structure specificcleavage assays (e.g., the “INVADER” flap assay, or invasive cleavageassay, (Hologic, Inc.) described, e.g., in U.S. Pat. Nos. 5,846,717,5,985,557, 5,994,069, 6,001,567, 6,090,543, and 6,872,816; Lyamichev etal., Nat. Biotech., 17:292 (1999), Hall et al., PNAS, USA, 97:8272(2000), and in combined PCR/invasive cleavage assays (Hologic, Inc.,e.g., in U.S. Patent Publications 2006/0147955 and 2009/0253142), eachof which is herein incorporated by reference in its entirety for allpurposes); enzyme mismatch cleavage methods (e.g., Variagenics, U.S.Pat. Nos. 6,110,684, 5,958,692, 5,851,770, herein incorporated byreference in their entireties); polymerase chain reaction (PCR),described above; branched hybridization methods (e.g., Chiron, U.S. Pat.Nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802, herein incorporatedby reference in their entireties); rolling circle replication (e.g.,U.S. Pat. Nos. 6,210,884, 6,183,960 and 6,235,502, herein incorporatedby reference in their entireties); NASBA (e.g., U.S. Pat. No. 5,409,818,herein incorporated by reference in its entirety); molecular beacontechnology (e.g., U.S. Pat. No. 6,150,097, herein incorporated byreference in its entirety); E-sensor technology (U.S. Pat. Nos.6,248,229, 6,221,583, 6,013,170, and 6,063,573, herein incorporated byreference in their entireties); cycling probe technology (e.g., U.S.Pat. Nos. 5,403,711, 5,011,769, and 5,660,988, herein incorporated byreference in their entireties); Dade Behring signal amplificationmethods (e.g., U.S. Pat. Nos. 6,121,001, 6,110,677, 5,914,230,5,882,867, and 5,792,614, herein incorporated by reference in theirentireties); ligase chain reaction (e.g., Barany Proc. Natl. Acad. SciUSA 88, 189-93 (1991)); and sandwich hybridization methods (e.g., U.S.Pat. No. 5,288,609, herein incorporated by reference in its entirety).

In some embodiments, target nucleic acid is amplified (e.g., by PCR) andamplified nucleic acid is detected simultaneously using an invasivecleavage assay. Assays configured for performing a detection assay(e.g., invasive cleavage assay) in combination with an amplificationassay are described in US Patent Publication 20090253142 A1 (applicationSer. No. 12/404,240), incorporated herein by reference in its entiretyfor all purposes, and as diagrammed in FIG. 1. Because many copies ofthe FRET cassette are cleaved for each copy of the target ampliconproduced, the assay is said to produce “signal amplification” inaddition to target amplification. Additional amplification plus invasivecleavage detection configurations, termed the QuARTS method, aredescribed in U.S. Pat. Nos. 8,361,720, 8,715,937, and 8,916,344,incorporated herein by reference in their entireties for all purposes.

As used herein, the term “PCR-flap assay” is used interchangeably withthe term “PCR-invasive cleavage assay” and refers to an assayconfiguration combining PCR target amplification and detection of theamplified DNA by formation of a first overlap cleavage structurecomprising amplified target DNA, and a second overlap cleavage structurecomprising a cleaved 5′ flap from the first overlap cleavage structureand a labeled hairpin detection oligonucleotide called a “FRETcassette”. In the PCR-flap assay as used herein, the assay reagentscomprise a mixture containing DNA polymerase, FEN-1 endonuclease, aprimary probe comprising a portion complementary to a target nucleicacid, and a hairpin FRET cassette, and the target nucleic acid isamplified by PCR and the amplified nucleic acid is detectedsimultaneously (i.e., detection occurs during the course of targetamplification). PCR-flap assays include the QuARTS assays described inU.S. Pat. Nos. 8,361,720; 8,715,937; and 8,916,344, and theamplification assays of U.S. Pat. No. 9,096,893 (for example, asdiagrammed in FIG. 1 of that patent), each of which is incorporatedherein by reference in its entirety.

As used herein, the term “PCR-flap assay reagents” refers to acollection of reagents for detecting target sequences in a PCR-flapassay, the reagents comprising nucleic acid molecules capable ofparticipating in amplification of a target nucleic acid and in formationof a flap cleavage structure in the presence of the target sequence, ina mixture containing DNA polymerase, FEN-1 endonuclease and a FRETcassette. PCR-flap assay reagents typically contain a forward primer, areverse primer, an invasive oligonucleotide, a flap oligonucleotide, apolymerase, a flap endonuclease and a FRET cassette. In someembodiments, the forward primer acts as a primer in the PCR reaction andas an invasive oligonucleotide in the cleavage reaction. In theseembodiments, the invasive oligonucleotide and the forward primer havethe same sequence.

As used herein, the term “flap assay reagents” or “invasive cleavageassay reagents” refers to all reagents that are required for performinga flap assay or invasive cleavage assay on a substrate. As is known inthe art, flap assays generally include an invasive oligonucleotide, aflap oligonucleotide, a flap endonuclease and a FRET cassette, asdescribed above. Flap assay reagents may optionally contain a target towhich the invasive oligonucleotide and flap oligonucleotide bind.

As used herein, the term “flap oligonucleotide” refers to anoligonucleotide that: (i) hybridizes to the target nucleic acid and (ii)is cleaved by a flap endonuclease in an invasive cleavage assay. Asshown in FIG. 1, a flap oligonucleotide has at least two regions: (i) atarget specific region (which may also be referred to as an analytespecific region or “ASR”; i.e., a sequence that hybridizes to thetarget, labeled as a “Specific Probe” in FIG. 1) and (ii) a “flap”,i.e., a sequence that is 5′ to the target specific region and does nothybridize with the target (labeled as a “Probe Arm” in FIG. 1). A flapoligonucleotide forms an invasive cleavage structure with the targetnucleic acid and the invasive oligonucleotide. As shown in FIG. 1, the“flap” of the flap oligonucleotide is cleaved off in an invasivecleavage reaction. In some embodiments, the flap may be at least 6 basesin length (e.g., 8-12 bases in length). In some embodiments, theanalyte-specific region of a flap oligonucleotide may be at least 13bases, e.g., 13-23 bases, 14-23 bases or 15-23 bases, in length.

As used herein, the term “FRET cassette” refers to a hairpinoligonucleotide that contains a fluorophore moiety and a nearby quenchermoiety that quenches the fluorophore. Hybridization of a cleaved flap(e.g., from cleavage of a target-specific probe in a PCR-flap assayassay) with a FRET cassette produces a secondary substrate for the flapendonuclease, e.g., a FEN-1 enzyme. Once this substrate is formed, the5′ fluorophore-containing base is cleaved from the cassette, therebygenerating a fluorescence signal. In preferred embodiments, a FRETcassette comprises an unpaired 3′ portion to which a cleavage product,e.g., a portion of a cleaved flap oligonucleotide, can hybridize to froman invasive cleavage structure cleavable by a FEN-1 endonuclease.

A nucleic acid “hairpin” as used herein refers to a region of asingle-stranded nucleic acid that contains a duplex (i.e., base-paired)stem and a loop, formed when the nucleic acid comprises two portionsthat are sufficiently complementary to each other to form a plurality ofconsecutive base pairs.

As used herein, the term “FRET” refers to fluorescence resonance energytransfer, a process in which moieties (e.g., fluorophores) transferenergy e.g., among themselves, or, from a fluorophore to anon-fluorophore (e.g., a quencher molecule). In some circumstances, FRETinvolves an excited donor fluorophore transferring energy to alower-energy acceptor fluorophore via a short-range (e.g., about 10 nmor less) dipole-dipole interaction. In other circumstances, FRETinvolves a loss of fluorescence energy from a donor and an increase influorescence in an acceptor fluorophore. In still other forms of FRET,energy can be exchanged from an excited donor flurophore to anon-fluorescing molecule (e.g., a quenching molecule). FRET is known tothose of skill in the art and has been described (See, e.g., Stryer etal., 1978, Ann. Rev. Biochem., 47:819; Selvin, 1995, Methods Enzymol.,246:300; Orpana, 2004 Biomol Eng 21, 45-50; Olivier, 2005 Mutant Res573, 103-110, each of which is incorporated herein by reference in itsentirety).

As used herein, the term “FEN-1” in reference to an enzyme refers to anon-polymerase flap endonuclease from a eukaryote or archaeal organism,as encoded by a FEN-1 gene. See, e.g., WO 02/070755, and Kaiser M. W.,et al. (1999) J. Biol. Chem., 274:21387, which are incorporated byreference herein in their entireties for all purposes.

As used herein, the term “FEN-1 activity” refers to any enzymaticactivity of a FEN-1 enzyme.

As used herein, the term “primer annealing” refers to conditions thatpermit oligonucleotide primers to hybridize to template nucleic acidstrands. Conditions for primer annealing vary with the length andsequence of the primer and are generally based upon the T_(m) that isdetermined or calculated for the primer. For example, an annealing stepin an amplification method that involves thermocycling involves reducingthe temperature after a heat denaturation step to a temperature based onthe T_(m) of the primer sequence, for a time sufficient to permit suchannealing.

As used herein, the term “amplifiable nucleic acid” is used in referenceto nucleic acids that may be amplified by any amplification method. Itis contemplated that “amplifiable nucleic acid” will usually comprise“sample template.”

The term “real time” as used herein in reference to detection of nucleicacid amplification or signal amplification refers to the detection ormeasurement of the accumulation of products or signal in the reactionwhile the reaction is in progress, e.g., during incubation or thermalcycling. Such detection or measurement may occur continuously, or it mayoccur at a plurality of discrete points during the progress of theamplification reaction, or it may be a combination. For example, in apolymerase chain reaction, detection (e.g., of fluorescence) may occurcontinuously during all or part of thermal cycling, or it may occurtransiently, at one or more points during one or more cycles. In someembodiments, real time detection of PCR is accomplished by determining alevel of fluorescence at the same point (e.g., a time point in thecycle, or temperature step in the cycle) in each of a plurality ofcycles, or in every cycle. Real time detection of amplification may alsobe referred to as detection “during” the amplification reaction.

As used herein, the term “abundance of nucleic acid” refers to theamount of a particular target nucleic acid sequence present in a sampleor aliquot. The amount is generally referred to in terms of mass (e.g.,μg), mass per unit of volume (e.g., μg/μL); copy number (e.g., 1000copies, 1 attomole), or copy number per unit of volume (e.g., 1000copies per mL, 1 attomole per μL). Abundance of a nucleic acid can alsobe expressed as an amount relative to the amount of a standard of knownconcentration or copy number. Measurement of abundance of a nucleic acidmay be on any basis understood by those of skill in the art as being asuitable quantitative representation of nucleic acid abundance,including physical density or the sample, optical density, refractiveproperty, staining properties, or on the basis of the intensity of adetectable label, e.g. a fluorescent label.

The term “amplicon” or “amplified product” refers to a segment ofnucleic acid, generally DNA, generated by an amplification process suchas the PCR process. The terms are also used in reference to RNA segmentsproduced by amplification methods that employ RNA polymerases, such asNASBA, TMA, etc.

The term “amplification plot” as used in reference to a thermal cyclingamplification reaction refers to the plot of signal that is indicativeof amplification, e.g., fluorescence signal, versus cycle number. Whenused in reference to a non-thermal cycling amplification method, anamplification plot generally refers to a plot of the accumulation ofsignal as a function of time.

The term “baseline” as used in reference to an amplification plot refersto the detected signal coming from assembled amplification reactionsprior to incubation or, in the case of PCR, in the initial cycles, inwhich there is little change in signal.

The term “C_(t)” or “threshold cycle” as used herein in reference toreal time detection during an amplification reaction that is thermalcycled refers to the fractional cycle number at which the detectedsignal (e.g., fluorescence) passes the fixed threshold.

The term “no template control” and “no target control” (or “NTC”) asused herein in reference to a control reaction refers to a reaction orsample that does not contain template or target nucleic acid. It is usedto verify amplification quality.

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample that is analyzed for the presence of “target.”In contrast, “background template” is used in reference to nucleic acidother than sample template that may or may not be present in a sample.The presence of background template is most often inadvertent. It may bethe result of carryover, or it may be due to the presence of nucleicacid contaminants sought to be purified away from the sample. Forexample, nucleic acids from organisms other than those to be detectedmay be present as background in a test sample.

A sample “suspected of containing” a nucleic acid may contain or notcontain the target nucleic acid molecule.

As used herein, the term “sample” is used in its broadest sense. Forexample, in some embodiments, it is meant to include a specimen orculture (e.g., microbiological culture), whereas in other embodiments,it is meant to include both biological and environmental samples (e.g.,suspected of comprising a target sequence, gene or template). In someembodiments, a sample may include a specimen of synthetic origin.Samples may be unpurifed or may be partially or completely purified orotherwise processed.

The present technology is not limited by the type of biological sampleused or analyzed. The present technology is useful with a variety ofbiological samples including, but not limited to, tissue (e.g., organ(e.g., heart, liver, brain, lung, stomach, intestine, spleen, kidney,pancreas, and reproductive organs), glandular, skin, and muscle), cell(e.g., blood cell (e.g., lymphocyte or erythrocyte), muscle cell, tumorcell, and skin cell), gas, bodily fluid (e.g., blood or portion thereof,serum, plasma, urine, semen, saliva, etc.), or solid (e.g., stool)samples obtained from a human (e.g., adult, infant, or embryo) or animal(e.g., cattle, poultry, mouse, rat, dog, pig, cat, horse, and the like).In some embodiments, biological samples may be solid food and/or feedproducts and/or ingredients such as dairy items, vegetables, meat andmeat by-products, and waste. Biological samples may be obtained from allof the various families of domestic animals, as well as feral or wildanimals, including, but not limited to, such animals as ungulates, bear,fish, lagomorphs, rodents, pinnipeds, etc.

Biological samples also include biopsies and tissue sections (e.g.,biopsy or section of tumor, growth, rash, infection, orparaffin-embedded sections), medical or hospital samples (e.g.,including, but not limited to, blood samples, saliva, buccal swab,cerebrospinal fluid, pleural fluid, milk, colostrum, lymph, sputum,vomitus, bile, semen, oocytes, cervical cells, amniotic fluid, urine,stool, hair, and sweat), laboratory samples (e.g., subcellularfractions), and forensic samples (e.g., blood or tissue (e.g., spatteror residue), hair and skin cells containing nucleic acids), andarcheological samples (e.g., fossilized organisms, tissue, or cells).

Environmental samples include, but are not limited to, environmentalmaterial such as surface matter, soil, water (e.g., freshwater orseawater), algae, lichens, geological samples, air containing materialscontaining nucleic acids, crystals, and industrial samples, as well assamples obtained from food and dairy processing instruments, apparatus,equipment, utensils, disposable and non-disposable items.

Samples may be prepared by any desired or suitable method. In someembodiments, nucleic acids are analyzed directly from bodily fluids,stool, or other samples using the methods described in U.S. Pat. No.9,000,146, which is herein incorporated by reference in its entirety forall purposes.

The above described examples are not, however, to be construed aslimiting the sample (e.g., suspected of comprising a target sequence,gene or template (e.g., the presence or absence of which can bedetermined using the compositions and methods of the presenttechnology)) types applicable to the present technology.

The terms “nucleic acid sequence” and “nucleic acid molecule” as usedherein refer to an oligonucleotide, nucleotide or polynucleotide, andfragments or portions thereof. The terms encompass sequences thatinclude analogs of DNA and RNA nucleotides, including those listedabove, and also including, but not limited to, 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxyl-methyl)uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxy-amino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine,2,6-diaminopurine, and pyrazolo[3,4-d]pyrimidines such as guanineanalogue 6 amino 1H-pyrazolo[3,4d]pyrimidin 4(5H) one (ppG or PPG, alsoSuper G) and the adenine analogue 4 amino 1H-pyrazolo[3,4d]pyrimidine(ppA or PPA). The xanthine analogue 1H-pyrazolo[5,4d]pyrimidin4(5H)-6(7H)-dione (ppX) can also be used. These base analogues, whenpresent in an oligonucleotide, strengthen hybridization and improvemismatch discrimination. All tautomeric forms of naturally-occurringbases, modified bases and base analogues may be included in theoligonucleotide conjugates of the technology. Other modified basesuseful in the present technology include6-amino-3-prop-1-ynyl-5-hydropyrazolo[3,4-d]pyrimidine-4-one, PPPG;6-amino-3-(3-hydroxyprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidine-4-one,HOPPPG;6-amino-3-(3-aminoprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidine-4-one,NH2PPPG; 4-amino-3-(prop-1-ynyl)pyrazolo[3,4-d]pyrimidine, PPPA;4-amino-3-(3-hydroxyprop-1-ynyl)pyrazolo[3,4-d]pyrimidine, HOPPPA;4-amino-3-(3-aminoprop-1-ynyl)pyrazolo[3,4-d]pyrimidine, NH₂ PPPA;3-prop-1-ynylpyrazolo[3,4-d]pyrimidine-4,6-diamino, (NH₂)₂ PPPA;2-(4,6-diaminopyrazolo[3,4-d]pyrimidin-3-yl)ethyn-1-ol, (NH₂)₂ PPPAOH;3-(2-aminoethynyl)pyrazolo[3,4-d]pyrimidine-4,6-diamine, (NH₂)₂ PPPANH₂;5-prop-1-ynyl-1,3-dihydropyrimidine-2,4-dione, PU;5-(3-hydroxyprop-1-ynyl)-1,3-dihydropyrimidine-2,4-dione, HOPU;6-amino-5-prop-1-ynyl-3-dihydropyrimidine-2-one, PC;6-amino-5-(3-hydroxyprop-1-yny)-1,3-dihydropyrimidine-2-one, HOPC; and6-amino-5-(3-aminoprop-1-yny)-1,3-dihydropyrimidine-2-one, NH₂PC;5-[4-amino-3-(3-methoxyprop-1-ynyl)pyrazol[3,4-d]pyrimidinyl]-2-(hydroxymethyl)oxolan-3-ol,CH₃ OPPPA;6-amino-1-[4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-3-(3-methoxyprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidin-4-one,CH₃ OPPPG; 4,(4,6-Diamino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-but-3-yn-1-ol, Super A;6-Amino-3-(4-hydroxy-but-1-ynyl)-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one;5-(4-hydroxy-but-1-ynyl)-1H-pyrimidine-2,4-dione, Super T;3-iodo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine ((NH₂)₂PPAI);3-bromo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine ((NH₂)₂ PPABr);3-chloro-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine ((NH₂)₂PPAC1);3-Iodo-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine (PPAI);3-Bromo-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine (PPABr); and3-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine (PPAC1).

A nucleic acid sequence or molecule may be DNA or RNA, of either genomicor synthetic origin, that may be single or double stranded, andrepresent the sense or antisense strand. Thus, nucleic acid sequence maybe dsDNA, ssDNA, mixed ssDNA, mixed dsDNA, dsDNA made into ssDNA (e.g.,through melting, denaturing, helicases, etc.), A-, B-, or Z-DNA,triple-stranded DNA, RNA, ssRNA, dsRNA, mixed ss and dsRNA, dsRNA madeinto ssRNA (e.g., via melting, denaturing, helicases, etc.), messengerRNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), catalytic RNA,snRNA, microRNA, or protein nucleic acid (PNA).

The present technology is not limited by the type or source of nucleicacid (e.g., sequence or molecule (e.g. target sequence and/oroligonucleotide)) utilized. For example, the nucleic acid sequence maybe amplified or created sequence (e.g., amplification or creation ofnucleic acid sequence via synthesis (e.g., polymerization (e.g., primerextension (e.g., RNA-DNA hybrid primer technology)) and reversetranscription (e.g., of RNA into DNA)) and/or amplification (e.g.,polymerase chain reaction (PCR), rolling circle amplification (RCA),nucleic acid sequence based amplification (NASBA), transcriptionmediated amplification (TMA), ligase chain reaction (LCR), cycling probetechnology, Q-beta replicase, strand displacement amplification (SDA),branched-DNA signal amplification (bDNA), hybrid capture, and helicasedependent amplification).

The terms “nucleotide” and “base” are used interchangeably when used inreference to a nucleic acid sequence, unless indicated otherwise herein.A “nucleobase” is a heterocyclic base such as adenine, guanine,cytosine, thymine, uracil, inosine, xanthine, hypoxanthine, or aheterocyclic derivative, analog, or tautomer thereof. A nucleobase canbe naturally occurring or synthetic. Non-limiting examples ofnucleobases are adenine, guanine, thymine, cytosine, uracil, xanthine,hypoxanthine, 8-azapurine, purines substituted at the 8 position withmethyl or bromine, 9-oxo-N6-methyladenine, 2-aminoadenine,7-deazaxanthine, 7-deazaguanine, 7-deaza-adenine, N4-ethanocytosine,2,6-diaminopurine, N6-ethano-2,6-diaminopurine, 5-methylcytosine,5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, thiouracil,pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine,isoguanine, inosine, 7,8-dimethylalloxazine, 6-dihydrothymine,5,6-dihydrouracil, 4-methyl-indole, ethenoadenine and the non-naturallyoccurring nucleobases described in U.S. Pat. Nos. 5,432,272 and6,150,510 and PCT applications WO 92/002258, WO 93/10820, WO 94/22892,and WO 94/24144, and Fasman (“Practical Handbook of Biochemistry andMolecular Biology”, pp. 385-394, 1989, CRC Press, Boca Raton, LO), allherein incorporated by reference in their entireties.

The term “oligonucleotide” as used herein is defined as a moleculecomprising two or more nucleotides (e.g., deoxyribonucleotides orribonucleotides), preferably at least 5 nucleotides, more preferably atleast about 10-15 nucleotides and more preferably at least about 15 to30 nucleotides, or longer (e.g., oligonucleotides are typically lessthan 200 residues long (e.g., between 15 and 100 nucleotides), however,as used herein, the term is also intended to encompass longerpolynucleotide chains). The exact size will depend on many factors,which in turn depend on the ultimate function or use of theoligonucleotide. Oligonucleotides are often referred to by their length.For example a 24 residue oligonucleotide is referred to as a “24-mer”.Oligonucleotides can form secondary and tertiary structures byself-hybridizing or by hybridizing to other polynucleotides. Suchstructures can include, but are not limited to, duplexes, hairpins,cruciforms, bends, and triplexes. Oligonucleotides may be generated inany manner, including chemical synthesis, DNA replication, reversetranscription, PCR, or a combination thereof. In some embodiments,oligonucleotides that form invasive cleavage structures are generated ina reaction (e.g., by extension of a primer in an enzymatic extensionreaction).

Because mononucleotides are reacted to make oligonucleotides in a mannersuch that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. A first regionalong a nucleic acid strand is said to be upstream of another region ifthe 3′ end of the first region is before the 5′ end of the second regionwhen moving along a strand of nucleic acid in a 5′ to 3′ direction.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (e.g., a sequence of two or morenucleotides (e.g., an oligonucleotide or a target nucleic acid)) relatedby the base-pairing rules. For example, the sequence “5′-A-G-T-3′,” iscomplementary to the sequence “3′-T-C-A-5′.” Complementarity may be“partial,” in which only some of the nucleic acid bases are matchedaccording to the base pairing rules. Or, there may be “complete” or“total” complementarity between the nucleic acid bases. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions, aswell as detection methods that depend upon the association of two ormore nucleic acid strands. Either term may also be used in reference toindividual nucleotides, especially within the context ofpolynucleotides. For example, a particular nucleotide within anoligonucleotide may be noted for its complementarity, or lack thereof,to a nucleotide within another nucleic acid sequence (e.g., a targetsequence), in contrast or comparison to the complementarity between therest of the oligonucleotide and the nucleic acid sequence.

The complement of a nucleic acid sequence as used herein refers to anoligonucleotide which, when aligned with the nucleic acid sequence suchthat the 5′ end of one sequence is paired with the 3′ end of the other,is in “antiparallel association.” Nucleotide analogs, as discussedabove, may be included in the nucleic acids of the present technologyand include. Complementarity need not be perfect; stable duplexes maycontain mismatched base pairs or unmatched bases. Those skilled in theart of nucleic acid technology can determine duplex stabilityempirically considering a number of variables including, for example,the length of the oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.

As used herein, the term “label” refers to any moiety (e.g., chemicalspecies) that can be detected or can lead to a detectable response. Insome preferred embodiments, detection of a label provides quantifiableinformation. Labels can be any known detectable moiety, such as, forexample, a radioactive label (e.g., radionuclides), a ligand (e.g.,biotin or avidin), a chromophore (e.g., a dye or particle that imparts adetectable color), a hapten (e.g., digoxygenin), a mass label, latexbeads, metal particles, a paramagnetic label, a luminescent compound(e.g., bioluminescent, phosphorescent or chemiluminescent labels) or afluorescent compound.

A label may be joined, directly or indirectly, to an oligonucleotide orother biological molecule. Direct labeling can occur through bonds orinteractions that link the label to the oligonucleotide, includingcovalent bonds or non-covalent interactions such as hydrogen bonding,hydrophobic and ionic interactions, or through formation of chelates orcoordination complexes. Indirect labeling can occur through use of abridging moiety or “linker”, such as an antibody or additionaloligonucleotide(s), which is/are either directly or indirectly labeled.

Labels can be used alone or in combination with moieties that cansuppress (e.g., quench), excite, or transfer (e.g., shift) emissionspectra (e.g., fluorescence resonance energy transfer (FRET)) of a label(e.g., a luminescent label).

A “polymerase” is an enzyme generally for joining 3′-OH 5′-triphosphatenucleotides, oligomers, and their analogs. Polymerases include, but arenot limited to, template-dependent DNA-dependent DNA polymerases,DNA-dependent RNA polymerases, RNA-dependent DNA polymerases, andRNA-dependent RNA polymerases. Polymerases include but are not limitedto T7 DNA polymerase, T3 DNA polymerase, T4 DNA polymerase, T7 RNApolymerase, T3 RNA polymerase, SP6 RNA polymerase, DNA polymerase 1,Klenow fragment, Thermophilus aquaticus DNA polymerase, Tth DNApolymerase, Vent DNA polymerase (New England Biolabs), Deep Vent DNApolymerase (New England Biolabs), Bst DNA Polymerase Large Fragment,Stoeffel Fragment, 9° N DNA Polymerase, Pfu DNA Polymerase, Tfl DNAPolymerase, RepliPHI Phi29 Polymerase, Tli DNA polymerase, eukaryoticDNA polymerase beta, telomerase, Therminator polymerase (New EnglandBiolabs), KOD HiFi DNA polymerase (Novagen), KOD1 DNA polymerase, Q-betareplicase, terminal transferase, AMV reverse transcriptase, M-MLVreverse transcriptase, Phi6 reverse transcriptase, HIV-1 reversetranscriptase, novel polymerases discovered by bioprospecting, andpolymerases cited in US 2007/0048748, U.S. Pat. Nos. 6,329,178;6,602,695; and U.S. Pat. No. 6,395,524 (incorporated by reference).These polymerases include wild-type, mutant isoforms, and geneticallyengineered variants.

A “DNA polymerase” is a polymerase that produces DNA fromdeoxynucleotide monomers (dNTPs). “Eubacterial DNA polymerase” as usedherein refers to the Pol A type DNA polymerases (repair polymerases)from Eubacteria, including but not limited to DNA Polymerase I from E.coli, Taq DNA polymerase from Thermus aquaticus and DNA Pol I enzymesfrom other members of genus Thermus, and other eubacterial species etc.

As used herein, the term “target” refers to a nucleic acid species ornucleic acid sequence or structure to be detected or characterized.

Accordingly, as used herein, “non-target”, e.g., as it is used todescribe a nucleic acid such as a DNA, refers to nucleic acid that maybe present in a reaction, but that is not the subject of detection orcharacterization by the reaction. In some embodiments, non-targetnucleic acid may refer to nucleic acid present in a sample that doesnot, e.g., contain a target sequence, while in some embodiments,non-target may refer to exogenous nucleic acid, i.e., nucleic acid thatdoes not originate from a sample containing or suspected of containing atarget nucleic acid, and that is added to a reaction, e.g., to normalizethe activity of an enzyme (e.g., polymerase) to reduce variability inthe performance of the enzyme in the reaction.

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleoside triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template, and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel.

As used herein, the term “control” when used in reference to nucleicacid detection or analysis refers to a nucleic acid having knownfeatures (e.g., known sequence, known copy-number per cell), for use incomparison to an experimental target (e.g., a nucleic acid of unknownconcentration). A control may be an endogenous, preferably invariantgene against which a test or target nucleic acid in an assay can benormalized. Such normalizing controls for sample-to-sample variationsthat may occur in, for example, sample processing, assay efficiency,etc., and allows accurate sample-to-sample data comparison. Genes thatfind use for normalizing nucleic acid detection assays on human samplesinclude, e.g., β-actin, ZDHHCl, and B3GALT6 (see, e.g., U.S. patentapplication Ser. Nos. 14/966,617 and 62/364,082, each incorporatedherein by reference.

Controls may also be external. For example, in quantitative assays suchas qPCR, QuARTS, etc., a “calibrator” or “calibration control” is anucleic acid of known sequence, e.g., having the same sequence as aportion of an experimental target nucleic acid, and a knownconcentration or series of concentrations (e.g., a serially dilutedcontrol target for generation of calibration curved in quantitativePCR). Typically, calibration controls are analyzed using the samereagents and reaction conditions as are used on an experimental DNA. Incertain embodiments, the measurement of the calibrators is done at thesame time, e.g., in the same thermal cycler, as the experimental assay.In preferred embodiments, multiple calibrators may be included in asingle plasmid, such that the different calibrator sequences are easilyprovided in equimolar amounts. In particularly preferred embodiments,plasmid calibrators are digested, e.g., with one or more restrictionenzymes, to release calibrator portion from the plasmid vector. See,e.g., WO 2015/066695, which is included herein by reference.

As used herein “ZDHHCl” refers to a gene encoding a proteincharacterized as a zinc finger, DHHC-type containing 1, located in humanDNA on Chr 16 (16q22.1) and belonging to the DHHC palmitoyltransferasefamily.

As used herein, the term “process control” refers to an exogenousmolecule, e.g., an exogenous nucleic acid added to a sample prior toextraction of target DNA that can be measured post-extraction to assessthe efficiency of the process and be able to determine success orfailure modes. The nature of the process control nucleic acid used isusually dependent on the assay type and the material that is beingmeasured. For example, if the assay being used is for detection and/orquantification of double stranded DNA or mutations in it, then doublestranded DNA process controls are typically spiked into the samplespre-extraction. Similarly, for assays that monitor mRNA or microRNAs,the process controls used are typically either RNA transcripts orsynthetic RNA. See, e.g., U.S. Pat. Appl. Ser. No. 62/364,049, filedJul. 19, 2016, which is incorporated herein by reference, and whichdescribes use of zebrafish DNA as a process control for human samples.

As used herein, the term “zebrafish DNA” is distinct from bulk “fishDNA”) e.g., purified salmon DNA) and refers to DNA isolated from Daniorerio, or created in vitro (e.g., enzymatically, synthetically) to havea sequence of nucleotides found in DNA from Danio rerio. In preferredembodiments, the zebrafish DNA is a methylated DNA added as a detectablecontrol DNA, e.g., a process control for verifying DNA recovery throughsample processing steps. In particular, zebrafish DNA comprising atleast a portion of the RASSF1 gene finds use as a process control, e.g.,for human samples, as described in U.S. Pat. Appl. Ser. No. 62/364,049.

As used herein the term “fish DNA” is distinct from zebrafish DNA andrefers to bulk (e.g., genomic) DNA isolated from fish, e.g., asdescribed in U.S. Pat. No. 9,212,392. Bulk purified fish DNA iscommercially available, e.g., provided in the form of cod and/or herringsperm DNA (Roche Applied Science, Mannheim, Germany) or salmon DNA(USB/Affymetrix).

As used herein, the terms “particle” and “beads” are usedinterchangeable, and the terms “magnetic particles” and “magnetic beads”are used interchangeably and refer to particles or beads that respond toa magnetic field. Typically, magnetic particles comprise materials thathave no magnetic field but that form a magnetic dipole when exposed to amagnetic field, e.g., materials capable of being magnetized in thepresence of a magnetic field but that are not themselves magnetic in theabsence of such a field. The term “magnetic” as used in this contextincludes materials that are paramagnetic or superparamagnetic materials.The term “magnetic”, as used herein, also encompasses temporarilymagnetic materials, such as ferromagnetic or ferrimagnetic materialswith low Curie temperatures, provided that such temporarily magneticmaterials are paramagnetic in the temperature range at which silicamagnetic particles containing such materials are used according to thepresent methods to isolate biological materials.

As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of nucleic acid purificationsystems and reaction assays, such delivery systems include systems thatallow for the storage, transport, or delivery of reagents and devices(e.g., chaotropic salts, particles, buffers, denaturants,oligonucleotides, filters etc. in the appropriate containers) and/orsupporting materials (e.g., sample processing or sample storage vessels,written instructions for performing a procedure, etc.) from one locationto another. For example, kits include one or more enclosures (e.g.,boxes) containing the relevant reaction reagents and/or supportingmaterials. As used herein, the term “fragmented kit” refers to adelivery system comprising two or more separate containers that eachcontains a subportion of the total kit components. The containers may bedelivered to the intended recipient together or separately. For example,a first container may contain an materials for sample collection and abuffer, while a second container contains capture oligonucleotides anddenaturant. The term “fragmented kit” is intended to encompass kitscontaining Analyte specific reagents (ASR's) regulated under section520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limitedthereto. Indeed, any delivery system comprising two or more separatecontainers that each contains a subportion of the total kit componentsare included in the term “fragmented kit.” In contrast, a “combined kit”refers to a delivery system containing all of the components of areaction assay in a single container (e.g., in a single box housing eachof the desired components). The term “kit” includes both fragmented andcombined kits.

The term “system” as used herein refers to a collection of articles foruse for a particular purpose. In some embodiments, the articles compriseinstructions for use, as information supplied on e.g., an article, onpaper, on recordable media (e.g., diskette, CD, flash drive, etc.). Insome embodiments, instructions direct a user to an online location,e.g., a website for viewing, hearing, and/or downloading instructions.In some embodiments, instructions or other information are provided asan application (“app”) for a mobile device.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein is technology relating to the amplification-baseddetection of nucleic acids and particularly, but not exclusively, tomethods for enriching low-DNA, bisulfite-converted samples for analysis.

Biological samples of interest may have vastly different amounts of DNAin them, and even if rich in bulk DNA, may have very low amounts of DNAsof interest, e.g., non-normal DNAs within a background of normal DNA, orhuman DNA in a background of microbial DNA (or vice versa). Tocompensate for a low concentration of target DNA, a large sample maysometimes be processed to collect sufficient DNA for a particular assay.However, when it is desirable to subject a sample with a lowconcentration of target DNA to a number of different assays in parallel,the necessary sample size may become prohibitively large. For example,circulating free DNA in plasma of a subject is typically very low, as itis continuously cleared from the bloodstream, mainly by the liver, andhas a half-life of only 10 to 15 minutes. The typical levels ofcirculating DNA are thus very low, e.g., for healthy individuals, aparticular segment of DNA, e.g., from a gene of interest, may be presentat about 1,500-2000 copies/mL, while a segment of DNA associated with atumor may be present at about 5000 copies/mL in a subject with a latestage cancer. Further, tumor-derived cfDNA in plasma is typicallyfragmented into short strands, e.g., of 200 or fewer base pairs (see,e.g., P. Jiang, et al., Proc. Natl Acad Sci. 112(11): E1317-E1325(2015), incorporated herein by reference in its entirety). Such smallDNAs are especially hard to purify because they can be lost duringtypical purification steps, e.g., through inefficiencies inprecipitation and/or DNA binding purification steps.

Recovery of the DNA from such blood fraction samples may capture 75%,but often much less is recovered. Thus, depending on the sensitivity ofthe particular assay for these targets, analysis of multiple DNA markersfrom plasma can require large amounts of plasma from a subject.Enrichment by targeted pre-amplification of specific target regions canincrease the number of markers that can be analyzed using the samestarting sample, i.e., without the need to collect correspondinglylarger samples (e.g., plasma or blood) from the subject.

Provided herein are embodiments of technologies for extraction of DNA,e.g., cell-free circulating DNA, from plasma samples. In preferredembodiments, the methods provided herein do not comprise organicextraction (e.g., phenol-chloroform extraction), alcohol precipitation,or use of columns, making the methods readily scalable and automatable.In particularly preferred embodiments, essentially the entire isolationprocedure—from plasma sample to bead-bound purified DNA ready forelution—is performed at room temperature.

Provided herein are embodiments of technologies for multiplexedpre-amplification particularly suited for analysis of target DNAs thatare in low abundance and/or that are fragmented in the samples in whichthey are found, and that have been treated with bisulfite reagent, e.g.,as described in Leontiou, et al., PLoS ONE 10(8): e0135058. doi:10.1371/journal.pone.0135058 (2015). In certain preferred embodiments,the bisulfite treatment comprises use of ammonium hydrogen sulfite, withdesulfonation preferably performed on support-bound DNA, as described inU.S. Pat. No. 9,315,853.

Embodiments of the Technology

1. Isolation of Circulating Cell-Free DNA from Plasma

Provided herein is technology related to isolation of fragmented DNAfrom samples, e.g., blood or plasma samples. In particular, providedherein is technology related to extraction of low-copy, small DNAs,e.g., less than about 200 base pairs in length, from plasma samples,using mixable particles, e.g., silica particles, to bind DNA. Methodsare provided herein using two different lysis reagents, added atdifferent times during the lysis treatment of the plasma sample, andusing a combination of two different wash buffers in the processing ofDNA bound to the particles. In preferred embodiments, the technologyprovided herein comprises addition of a bulk exogenous non-target DNA,e.g., bulk fish DNA, to the DNA to be isolated for further analysis,preferably added to the plasma prior to or at the first particle-bindingstep.

2. Pre-Amplification of Target Regions for PCR-Flap Assay Analysis

Provided herein is technology related to providing an increased amountof DNA for analysis in a PCR-flap assay, e.g., a QuARTS assay asdiagramed in FIG. 1. In particular, embodiments of the methods andcompositions disclosed herein provide for increasing an amount of a DNAtarget of interest, e.g., from a low-target sample, using a multiplexedpre-amplification step, followed by target-specific detection to furtheramplify and to detect the target locus of interest.

Re-amplifying DNA segments previously amplified in a targeted manner,e.g., amplification of an aliquot or dilution of the amplicon product ofa target-specific PCR, is known to be prone to undesirable artifacts,e.g., high background of undesired DNA products. Thus, analysis oftarget nucleic acids using sequential rounds of specific PCR istypically conducted under special conditions, e.g., using differentprimers pairs in the sequential reactions. For example, in “nested PCR”the first round of amplification is conducted to produce a firstamplicon, and the second round of amplification is conducted using aprimer pair in which one or both of the primers anneal to sites insidethe regions defined by the initial primer pair, i.e., the second primerpair is considered to be “nested” within the first primer pair. In thisway, background amplification products from the first PCR that do notcontain the correct inner sequence are not further amplified in thesecond reaction. Other strategies to reduce undesirable effects includeusing very low concentrations of primers in the first amplification.

Multiplex amplification of a plurality of different specific targetsequences is typically conducted using relatively standard PCR reagentmixtures, e.g., for Amplitaq DNA polymerase, mixtures comprising 50 mMKCl, 1.5 to 2.5 mM MgCl₂, and Tris-HCl buffer at about pH 8.5 are used.As discussed above, if a second amplification is to be performed, theprimers are typically present in limited amounts (Andersson, supra). Fora subsequent assay, the amplified DNA is diluted or purified, and asmall aliquot is then added to a detection assay, e.g., a PCR-flapassay, which uses different buffer and salt conditions than standard PCR(e.g., a buffer comprising MOPS, Tris-HCl pH 8.0, and 7.5 mM MgCl₂, andlittle or no added KCl or other monovalent salt, conditions typicallyconsidered unfavorable for PCR due to the low monovalent salt and therelatively high concentration of Mg⁺⁺ (see, e.g., “Guidelines for PCROptimization with Taq DNA Polymerase”https://www.neb.com/tools-and-resources/usage-guidelines/guidelines-for-pcr-optimization-with-taq-dna-polymerase,which discloses 1.5 mM to 2.0 mM as the optimal Mg⁺⁺ range for Taq DNApolymerase, with optimization to be conducted by supplementing themagnesium concentration in 0.5 increments up to 4 mM. See also“Multiplex PCR: Critical Parameters and Step-by-Step Protocol” O.Henegariu, et al., BioTechniques 23:504-511 (September 1997). A changein reaction conditions between a first amplification and a secondamplification (or other detection assay) is often effected by eitherpurifying the DNA from the first amplification reaction or by usingsufficient dilution such that the amounts of reaction components carriedinto the follow-on reaction is negligible.

Embodiments of the present technology are directed to combiningbisulfite modification, multiplex PCR amplification, and PCR-flap assaydetection for the detection of low-copy number DNAs. During developmentof embodiments of the technology provided herein, it was discovered thatuse of a PCR-flap assay buffer with very low KCl and comprising elevatedMg⁺⁺ (e.g., >6 mM, preferably >7 mM, more preferably 7.5 mM), for boththe multiplex pre-amplification in the absence of the flap assayreagents (e.g., in the absence of the hairpin oligonucleotide and FEN-1endonuclease) and for the following PCR-flap assay producedsubstantially better signal. Further, it was unexpectedly determinedthat using the same primer pair to amplify a target region in both thepre-amplification and in the subsequent PCR-flap assay reaction producedbetter results than using a nested arrangement of primers. Use of thePCR-flap assay primers pairs in the initial amplification and in thePCR-flap assay has the advantage of producing signal from very smallfragments of target DNA, such as would be expected in remote DNAsamples. For example, amplicons of about 50 to 85 base pairs areproduced and detected in examples hereinbelow).

In some embodiments, the one or both of the pre-amplification and thePCR-flap assay comprise exogenous, non-target DNA in the reactionmixture, as described, e.g., in U.S. patent application Ser. No.14/036,649, filed Sep. 25, 2013, which is incorporated herein byreference in its entirety. In certain preferred embodiments, theexogenous non-target DNA comprises fish DNA. While not limiting theinvention to any particular mechanism of action, it has been observedthat the presence of hairpin oligonucleotides (e.g., hairpin FRETcassettes as used, for example, in some embodiments of invasive cleavagedetection assays) may have an inhibiting effect on DNA polymerasepresent in the same vessel, as assessed by sample and signalamplification. See, e.g., U.S. Patent Publication 2006/0147955 toAllawi, which is incorporated herein by reference for all purposes.Allawi et al. observed that when PCR and invasive cleavage assaycomponents were combined, the hairpin FRET oligonucleotides affectedpolymerase performance, and the use of purified exogenous non-targetDNA, especially genomic DNA, improves the consistency of signal producedin such assays. Thus, in preferred embodiments, purified exogenousnon-target DNA is added to samples before and/or while contacting thesamples with an enzyme such as a polymerase. The non-target DNA istypically added to the sample or reaction mixture, for example, at aconcentration of approximately 2 to 20 ng per μl of reaction mixture,preferably approximately 6 to approximately 7 ng per μl of reactionmixture, when approximately 0.01 to 1.0 U/μL of enzyme, e.g., 0.05 U/μLof enzyme (e.g., a polymerase such as, e.g., Taq polymerase) is used inthe assay.

Embodiments of the multiplex pre-amplification as disclosed herein finduse with PCR-flap assays such as the QuARTS assay. As diagrammed in FIG.1, the QuARTS technology combines a polymerase-based target DNAamplification process with an invasive cleavage-based signalamplification process. Fluorescence signal generated by the QuARTSreaction is monitored in a fashion similar to real-time PCR. During eachamplification cycle, three sequential chemical reactions occur in eachassay well, with the first and second reactions occurring on target DNAtemplates and the third occurring on a synthetic DNA target labeled witha fluorophore and quencher dyes, thus forming a fluorescence resonanceenergy transfer (FRET) donor and acceptor pair. The first reactionproduces amplified target with a polymerase and oligonucleotide primers,and the second reaction uses a highly structure-specific 5′-flapendonuclease-1 (FEN-1) enzyme reaction to release a 5′-flap sequencefrom a target-specific flap oligonucleotide that binds to the product ofthe polymerase reaction, forming an overlap flap substrate. In the thirdreaction, the cleaved flap anneals to a specially designedoligonucleotide containing a fluorophore and quencher closely linked ina FRET pair such that the fluorescence is quenched (FRET cassette). Thereleased probe flap hybridizes in a manner that forms an overlap flapsubstrate that allows the FEN-1 enzyme to cleave the 5′-flap containingthe fluorophore, thus releasing it from proximity to the quenchermolecule. The released fluorophore generates fluorescence signal to bedetected. During the second and third reactions, the FEN-1 endonucleasecan cut multiple probes per target, generating multiple cleaved 5′-flapsper target, and each cleaved 5′ flap can participate in the cleavage ofmany FRET cassettes, giving rise to additional fluorescence signalamplification in the overall reaction.

In some configurations, each assay is designed to detect multiple genes,e.g., 3 genes reporting to 3 distinct fluorescent dyes. See, e.g., Zou,et al., (2012) “Quantification of Methylated Markers with a MultiplexMethylation-Specific Technology”, Clinical Chemistry 58: 2, incorporatedherein by reference for all purposes.

3. Use of Flap Oligonucleotides that have a “Long” Target-SpecificRegion

How PCR-flap assays are performed at a molecular level is describedabove. As described above and shown in FIG. 1, PCR-flap assays employ anoligonucleotide (referred to as a “flap oligonucleotide”) thathybridizes to the target nucleic acid at a site that overlaps with thesite to which the invasive oligonucleotide binds. Together, the targetnucleic acid, invasive oligonucleotide and flap oligonucleotide form aninvasive cleavage structure that is cleaved by the flap endonuclease.Cleavage of this structure by the flap endonuclease releases theunhybridized 5′ tail of the flap oligonucleotide. As shown in FIG. 1, aflap oligonucleotide has at least two regions: a target specific regionthat hybridizes to the target and a “flap” sequence that is 5′ to thetarget specific region and does not hybridize with the target. In aninvasive cleavage reaction, the “flap” is cleaved off the flapoligonucleotide by a flap endonuclease.

Conventional PCR-flap assays typically employ flap oligonucleotides thathave a target-specific region that is no more than 12 bases in length(see, e.g., US20170121757, U.S. Pat. Nos. 9,096,893 and 8,715,937). Indeveloping the present technology, the inventors found that PCR-flapassays that employ flap oligonucleotides that have a “longer”target-specific region, e.g., a target-specific region of at least 13bases in length (e.g., in the range of 13 to 30 bases, 14 to 30 bases,or 15 to 30 bases in length), work just as well as, or in many caseseven better than, PCR-flap assays that employ flap oligonucleotides havea target-specific region of 12 bases in length.

PCR-flap assays that employ flap oligonucleotides have a target-specificregion of 12 bases in length can be implemented in at least two ways.For example, in some embodiments of the method the target-specificregion of a flap oligonucleotide can be extended to be at least 13 basesin length and one or more bases of the target-specific region can besubstituted with an inosine. Inosine pairs with any natural base. Inthese embodiments, the PCR-flap assay may be done using thermocyclingconditions that have already been optimized for flap oligonucleotideshaving a target-specific region that is 12 or less bases. For example,in these embodiments the reaction may be subjected to multiple cycles ofthe following steps: a denaturation step at least 90° C., an annealingstep at a temperature that is below 55° C., e.g., in the range of 50° C.to 55° C., and an extension step at a temperature in the range of 65° C.to 75° C. In other embodiments of the method, the target-specific regionof a flap oligonucleotide can be extended to be at least 13 bases inlength without making any base substitutions. In these embodiments, theentire target-specific region of the flap oligonucleotide may beperfectly complementary with the target. In these embodiments thereaction may be implemented using thermocycling conditions that have ahigher “annealing” temperature, e.g., by subjecting the reaction tomultiple cycles of the following steps: a denaturation step at least 90°C., an annealing step at a temperature that is at least 60° C., e.g., inthe range of 60° C. to 70° C. or 60° C. to 65° C., and an extension stepat a temperature in the range of 65° C. to 75° C.

The following publications are incorporated by reference herein fortheir disclosure of alternative genes/loci that can be assayed usingPCR-flap methods, probe designs for the same, assay conditions andanalyses methods: U.S. application Ser. No. 15/694,300 filed on Sep. 1,2017, PCT/US17/42902, U.S. application Ser. No. 15/471,337 and PCT/US17/42902.

These embodiments are further illustrated by the examples providedbelow.

EXPERIMENTAL EXAMPLES Example 1 DNA Isolation from Cells and Plasma andBisulfite Conversion DNA Isolation

For cell lines, genomic DNA was isolated from cell-conditioned mediausing the “Maxwell® RSC ccfDNA Plasma Kit (Promega Corp., Madison,Wis.). Following the kit protocol, 1 mL of cell-conditioned media (CCM)is used in place of plasma, and processed according to the kitprocedure. The elution volume is 100 μL, of which 70 μL are used forbisulfite conversion.

An exemplary procedure for isolating DNA from a 4 mL sample of plasmawould be conducted as follows:

-   -   To a 4 mL sample of plasma, 300 μL of proteinase K (20 mg/mL) is        added and mixed.    -   Add 3 μL of 1 μg/μL of Fish DNA to the plasma-proteinase K        mixture.    -   Add 2 mL of plasma lysis buffer to plasma.        -   Plasma lysis buffer is:            -   4.3M guanidine thiocyanate            -   10% IGEPAL CA-630 (Octylphenoxy                poly(ethyleneoxy)ethanol, branched) (5.3 g of IGEPAL                CA-630 combined with 45 mL of 4.8 M guanidine                thiocyanate)    -   Incubate mixtures at 55° C. for 1 hour with shaking at 500 rpm.    -   Add 3 mL of plasma lysis buffer and mix.    -   Add 200 μL magnetic silica binding beads [16 μg of beads/μL] and        mix again.    -   Add 2 mL of 100% isopropanol and mix.    -   Incubate at 30° C. for 30 minutes with shaking at 500 rpm.    -   Place tube(s) on magnet and let the beads collect. Aspirate and        discard the supernatant.    -   Add 750 μL guanidine hydrochloride-ethyl alcohol (GuHCl-EtOH)        wash solution to vessel containing the binding beads and mix.        -   GuHCl-EtOH wash solution is:            -   3M GuHCl            -   57% EtOH.    -   Shake at 400 rpm for 1 minute.    -   Transfer samples to a deep well plate or 2 mL microfuge tubes.    -   Place tubes on magnet and let the beads collect for 10 minutes.        Aspirate and discard the supernatant.    -   Add 1000 μL wash buffer (10 mM Tris HCl, 80% EtOH) to the beads,        and incubate at 30° C. for 3 minutes with shaking.    -   Place tubes on magnet and let the beads collect. Aspirate and        discard the supernatant.    -   Add 500 μL wash buffer to the beads and incubate at 30° C. for 3        minutes with shaking.    -   Place tubes on magnet and let the beads collect. Aspirate and        discard the supernatant.    -   Add 250 μL wash buffer and incubate at 30° C. for 3 minutes with        shaking.    -   Place tubes on magnet and let the beads collect. Aspirate and        discard the remaining buffer.    -   Add 250 μL wash buffer and incubate at 30° C. for 3 minutes with        shaking.    -   Place tubes on magnet and let the beads collect. Aspirate and        discard the remaining buffer.    -   Dry the beads at 70° C. for 15 minutes, with shaking.    -   Add 125 μL elution buffer (10 mM Tris HCl, pH 8.0, 0.1 mM EDTA)        to the beads and incubate at 65° C. for 25 minutes with shaking.    -   Place tubes on magnet and let the beads collect for 10 minutes.    -   Aspirate and transfer the supernatant containing the DNA to a        new vessel or tube.

Bisulfite Conversion I. Sulfonation of DNA Using Ammonium HydrogenSulfite

-   -   1. In each tube, combine 64 μL DNA, 7 μL 1 N NaOH, and 9 μL of        carrier solution containing 0.2 mg/mL BSA and 0.25 mg/mL of fish        DNA.    -   2. Incubate at 42° C. for 20 minutes.    -   3. Add 120 μL of 45% ammonium hydrogen sulfite and incubate at        660 for 75 minutes.    -   4. Incubate at 4° C. for 10 minutes.

II. Desulfonation Using Magnetic Beads Materials

Magnetic beads (Promega MagneSil Paramagnetic Particles, Promegacatalogue number AS1050, 16 μg/μL).

Binding buffer: 6.5-7 M guanidine hydrochoride.

Post-conversion Wash buffer: 80% ethanol with 10 mM Tris HCl (pH 8.0).

Desulfonation buffer: 70% isopropyl alcohol, 0.1 N NaOH was selected forthe desulfonation buffer.

Samples are mixed using any appropriate device or technology to mix orincubate samples at the temperatures and mixing speeds essentially asdescribed below. For example, a Thermomixer (Eppendorf) can be used forthe mixing or incubation of samples. An exemplary desulfonation is asfollows:

-   -   1. Mix bead stock thoroughly by vortexing bottle for 1 minute.    -   2. Aliquot 50 μL of beads into a 2.0 mL tube (e.g., from USA        Scientific).    -   3. Add 750 μL of binding buffer to the beads.    -   4. Add 150 μL of sulfonated DNA from step I.    -   5. Mix (e.g., 1000 RPM at 30° C. for 30 minutes).    -   6. Place tube on the magnet stand and leave in place for 5        minutes. With the tubes on the stand, remove and discard the        supernatant.    -   7. Add 1,000 μL of wash buffer. Mix (e.g., 1000 RPM at 30° C.        for 3 minutes).    -   8. Place tube on the magnet stand and leave in place for 5        minutes. With the tubes on the stand, remove and discard the        supernatant.    -   9. Add 250 μL of wash buffer. Mix (e.g., 1000 RPM at 30° C. for        3 minutes).    -   10. Place tube on magnetic rack; remove and discard supernatant        after 1 minute.    -   11. Add 200 μL of desulfonation buffer. Mix (e.g., 1000 RPM at        30° C. for 5 minutes).    -   12. Place tube on magnetic rack; remove and discard supernatant        after 1 minute.    -   13. Add 250 μL of wash buffer. Mix (e.g., 1000 RPM at 30° C. for        3 minutes).    -   14. Place tube on magnetic rack; remove and discard supernatant        after 1 minute.    -   15. Add 250 μL of wash buffer to the tube. Mix (e.g., 1000 RPM        at 30° C. for 3 minutes).    -   16. Place tube on magnetic rack; remove and discard supernatant        after 1 minute.    -   17. Incubate all tubes at 30° C. with the lid open for 15        minutes.    -   18. Remove tube from magnetic rack and add 70 μL of elution        buffer directly to the beads.    -   19. Incubate the beads with elution-buffer (e.g., 1000 RPM at        40° C. for 45 minutes).    -   20. Place tubes on magnetic rack for about one minute; remove        and save the supernatant.

The converted DNA is then used in pre-amplification and/or flapendonuclease assays, as described below.

Example 2 Multiplex Pre-Amplification—Cycles of Pre-Amplification

Using a nested approach, the effect of the number of PCR cycles wasexamined by conducting 5, 7 or 10 cycles using the outer primer pairsfor each target sample. The PCR-flap assays using inner primers wereused to further amplify and to analyze the pre-amplified products.

Experimental Conditions:

-   -   1. Sample source: DNA extracted from HCT116 cell lines and        bisulfite treated as described above;    -   2. 50 μL pre-amplification PCR reactions.    -   3. Targets regions tested: NDRG4, BMP3, SFMBT2, VAV3, ZDHHCl,        and 3-actin (see FIG. 5)    -   4. Reaction conditions used for both pre-amplification PCR and        the PCR-flap assay:        -   7.5 mM MgCl₂,        -   10 mM MOPS,        -   0.3 mM Tris-HCl, pH 8.0,        -   0.8 mM KCl,        -   0.1 μg/μl BSA,        -   0.0001% Tween-20,        -   0.0001% IGEPAL CA-630,        -   250 μM dNTP)        -   GoTaq polymerase at 0.025 U/μl (Promega Corp., Madison,            Wis.)        -   Primer pairs for bisulfite-converted NDRG4, BMP3, SFMBT2,            VAV3, ZDHHCl, and β-actin, as shown in FIGS. 5A-5F, at 500            nM each primer in both the pre-amplification and the            PCR-flap assay.

10 μL of prepared bisulfite-treated target DNA are used in each 50 μLPCR reaction. Pre-amplification cycling was as shown below:

Pre-Amplification Reaction Cycles: Stage Temp/Time #of CyclesPre-incubation 95° C./5′   1 Amplification 1 95° C./30″ varying 68°C./30″ 72° C./30″ Cooling 40° C./30″ 1

After PCR, 10 μL of the amplification reaction was diluted to 100 μL in10 mM Tris, 0.1 mM EDTA, and 10 μL of the diluted amplification productare used in a standard PCR-flap assay, as described below. Comparativeassays used a QuARTS PCR-flap assay directly on the bisulfite-treatedDNA, without pre-amplification.

An exemplary QuARTS reaction typically comprises approximately 400-600nM (e.g., 500 nM) of each primer and detection probe, approximately 100nM of the invasive oligonucleotide, approximately 600-700 nM of each FAM(e.g., as supplied commercially by Hologic), HEX (e.g., as suppliedcommercially by BioSearch Technologies, IDT), and Quasar 670 (e.g., assupplied commercially by BioSearch Technologies) FRET cassettes, 6.675ng/μL FEN-1 endonuclease (e.g., Cleavase® 2.0, Hologic, Inc.), 1 unitTaq DNA polymerase in a 30 μl reaction volume (e.g., GoTaq® DNApolymerase, Promega Corp., Madison, Wis.), 10 mM 3-(n-morpholino)propanesulfonic acid (MOPS), 7.5 mM MgCl₂, and 250 μM of each dNTP.

Exemplary QuARTS cycling conditions are as shown below:

QuARTS Reaction Cycle: Stage Temp/Time Number of Cycles AcquisitionPre-incubation 95° C./3′   1 none Amplification 1 95° C./20″ 10 none 63°C./30″ none 70° C./30″ none Amplification 2 95° C./20″ 35 none 53°C./1′   single 70° C./30″ none Cooling 40° C./30″ 1 none

The data are shown in FIG. 6, and show that 10 cycles ofpre-amplification gave the most consistent determination of thepercentage of methylation, as compared to the PCR-flap assay performedwithout pre-amplification.

Example 3 Nested Primers Vs. Non-Nested Primers; PCR Buffer Vs. PCR-FlapAssay Buffer

Assays were conducted to compare using a nested primer arrangement tothe use of the same PCR flap assay primers in both the pre-amplificationand the PCR-flap assay steps, and to compare the use of a typical PCRbuffer vs. a PCR-flap assay buffer during the pre-amplification step.The PCR-flap assay buffer was used. The typical PCR buffer was 1.5 mMMgCl₂, 20 mM Tris-HCl, pH 8, 50 mM KCl, 250 μM each dNTP; and thePCR-flap assay buffer was 7.5 mM MgCl₂, 10 mM MOPS, 0.3 mM Tris-HCl, pH8.0, 0.8 mM KCl, 0.1 μg/μL BSA, 0.0001% Tween-20, 0.0001% IGEPAL CA-630,250 μM each dNTP. Primer concentrations of 20 nM, 100 nM and 500 nM eachprimer were also compared.

Experimental Conditions:

-   -   1. Sample source: DNA extracted from HCT116 cell lines and        bisulfite treated;    -   2. 50 μL PCR reactions.    -   3. Targets regions tested: NDRG4, BMP3, SFMBT2, VAV3, ZDHHCl,        and 3-actin    -   4. GoTaq polymerase at 0.025 U/μL.    -   5. PCR or PCR-flap assay buffer, as described above,    -   6. Primer pairs for bisulfite-converted NDRG4, BMP3, SFMBT2,        VAV3, ZDHHCl, and β-actin, as shown in FIGS. 5A-5F, at 20 nM,        100 nM and 500 nM each primer.

Pre-amplification cycling was as shown below:

Pre-Amplification Reaction Cycle: Stage Temp/Time #of CyclesPre-incubation 95° C./5′ 1 Amplification 1 95° C./30″ 11 68° C./30″ 72°C./30″ Cooling 40° C./30″ 1

10 μL of prepared bisulfite-treated target DNA were used in each 50 μLPCR reaction. After PCR, 10 μL of the pre-amplification reaction wasdiluted to 100 μL in 10 mM Tris, 0.1 mM EDTA, and 10 μL of the dilutedamplification product are used in a standard PCR-flap assay, asdescribed in Example 2.

The data are shown in FIG. 7. The top panel shows expected yieldscalculated from starting DNA amounts and the second panel shows amountsdetected using the primer and buffer conditions indicated. These datashow that the highest nM concentrations of primers gave the highestamplification efficiency. Surprisingly, the PCR-flap assay buffer havingrelatively high Mg⁺⁺ and low KCl (7.5 mM 0.8 mM, respectively), whenused in the PCR pre-amplification, gave better results than use of atraditional PCR buffer having lower Mg⁺⁺ and much higher KClconcentration (1.5 mM and 50 mM, respectively). Further, using thePCR-flap assay primers (the “inner” primers and shown in FIGS. 5A-5F) inthe pre-amplification PCR worked as well or better than using sets outerand inner primer pairs in a nested PCR arrangement.

Example 4 Testing Cycles of Pre-Amplification in Flap Assay Buffer

Assays were conducted to determine effect of increasing the number ofpre-amplification PCR cycles on background in both no target controlsamples and on samples containing target DNA.

Experimental Conditions:

-   -   1. Sample source:        -   i) No target control=20 ng/μL fish DNA and/or 10 mM Tris,            0.1 mM EDTA;        -   ii) Bisulfite-converted DNA isolated from plasma from a            normal patient        -   iii) Bisulfite-converted DNA isolated from plasma from a            normal patient combined with DNA extracted from HCT116 cell            lines and bisulfite treated    -   2. 50 μL PCR reactions,    -   3. Targets regions tested: NDRG4, BMP3, SFMBT2, VAV3, ZDHHCl,        and β-actin,    -   4. Reaction conditions used for both pre-amplification and        PCR-flap assay:        -   7.5 mM MgCl₂,        -   10 mM MOPS,        -   0.3 mM Tris-HCl, pH 8.0,        -   0.8 mM KCl,        -   0.1 μg/μL BSA,        -   0.0001% Tween-20,        -   0.0001% IGEPAL CA-630,        -   250 μM dNTP)        -   GoTaq polymerase at 0.025 U/μl,        -   Primer pairs for bisulfite-converted NDRG4, BMP3, SFMBT2,            VAV3, ZDHHCl, and β-actin, as shown in FIGS. 5A-5F, at 500            nM each primer.

Pre-amplification cycling was as shown below:

Pre-Amplification Reaction Cycle: Stage Temp/Time #of CyclesPre-incubation 95° C./5′ 1 Amplification 1 95° C./30″ 5, 10, 20, or 2568° C./30″ 72° C./30″ Cooling 40° C./30″ 1

After PCR, 10 μL of the amplification reaction was diluted to 100 μL in10 mM Tris, 0.1 mM EDTA, and 10 μL of the diluted amplification productare used in a standard PCR-flap assay, as described in Example 1.

The data are shown in FIGS. 8A-8C, and showed that no background wasproduced in the no-target control reactions, even at the highest cyclenumber. However, the samples pre-amplified for 20 or 25 cycles showed anoticeable decrease in signal in the PCR-flap assay.

Example 5 Multiplex Targeted Pre-Amplification of Large-VolumeBisulfite-Converted DNA

To pre-amplify most or all of the bisulfite treated DNA from an inputsample, a large volume of the treated DNA may be used in a single,large-volume multiplex amplification reaction. For example, DNA isextracted from a cell lines (e.g., DFCI032 cell line (adenocarcinoma);H1755 cell line (neuroendocrine), using, for example, the MaxwellPromega blood kit # AS1400, as described above. The DNA is bisulfiteconverted, e.g., as described in Example 1.

A pre-amplification is conducted in a reaction mixture containing 7.5 mMMgCl₂, 10 mM MOPS, 0.3 mM Tris-HCl, pH 8.0, 0.8 mM KCl, 0.1 μg/μL BSA,0.0001% Tween-20, 0. 0001% IGEPAL CA-630, 250 μM dNTP, (e.g., 12 primerpairs/24 primers, in equimolar amounts, or with individual primerconcentrations adjusted to balance amplification efficiencies of thedifferent target regions), 0.025 units/μL HotStart GoTaq concentration,and 20 to 50% by volume of bisulfite-treated target DNA (e.g., 10 μL oftarget DNA into a 50 μL reaction mixture, or 50 μL of target DNA into a125 μL reaction mixture). Thermal cycling times and temperatures areselected to be appropriate for the volume of the reaction and theamplification vessel. For example, the reactions may be cycled asfollows

Stage Temp/Time #of Cycles Pre-incubation 95° C./5′ 1 Amplification 195° C./30″ 10 64° C./30″ 72° C./30″ Cooling  4° C./Hold 1After thermal cycling, aliquots of the pre-amplification reaction (e.g.,10 μL) are diluted to 500 μL in 10 mM Tris, 0.1 mM EDTA. Aliquots of thediluted pre-amplified DNA (e.g., 10 μL) are used in a QuARTS PCR-flapassay, e.g., as described in Example 2.

Example 6 Multiplex Targeted Pre-Amplification of Bisulfite-ConvertedDNA from Stool Samples

The multiplex pre-amplification methods described above were tested onDNA isolated from human stool samples.

Sample Source:

-   -   i) 4 DNA samples captured from stool samples (see, e.g., U.S.        Pat. No. 9,000,146) and bisulfite-treated according to Example        1, above, the samples having the following pathologies:

500237 Adenoma (AA) 500621 Adenocarcinoma (ACA) 780116 Normal 780687Normal

-   -   ii) No target control=20 ng/μL bulk fish DNA and/or 10 mM Tris,        0.1 mM EDTA;    -   2. 50 μL PCR reactions,    -   3. Targets regions tested: NDRG4, BMP3, SFMBT2, VAV3, ZDHHCl,        and 3-actin,    -   4. Reaction conditions used for both pre-amplification and        PCR-flap assay:        -   7.5 mM MgCl2,        -   10 mM MOPS,        -   0.3 mM Tris-HCl, pH 8.0,        -   0.8 mM KCl,        -   0.1 μg/μL BSA,        -   0.0001% Tween-20,        -   0.0001% IGEPAL CA-630,        -   250 μM dNTP)        -   GoTaq polymerase at 0.025 U/μl,        -   Primer pairs for bisulfite-converted NDRG4, BMP3, SFMBT2,            VAV3, ZDHHCl, and β-actin, as shown in FIGS. 5A-5F, at 500            nM each primer.

Pre-amplification cycling was as shown below:

Pre-Amplification Reaction Cycle: Stage Temp/Time #of CyclesPre-incubation 95° C./5′ 1 Amplification 1 95° C./30″ 10 68° C./30″ 72°C./30″ Cooling 40° C./30″ 1

After PCR, 10 μL of the amplification reaction was diluted to 100 μL in10 mM Tris, 0.1 mM EDTA, and 10 μL of the diluted amplification productare used in a standard PCR-flap assay, as described in Example 2.

The data are shown in FIG. 9, and show that no background was producedin the no-target control reactions. For samples in which the targetmarkers are not expected to be methylated (normal samples) no signal formethylated markers was detected, while the percent methylation detectedin the samples from subjects having adenoma or adenocarcinoma wereconsistent with the results obtained using a standard non-multiplexedQuARTS PCR-flap assay, i.e., without a separate pre-amplification step.

Example 7 Multiplex Targeted Pre-Amplification of Bisulfite-ConvertedDNA from Plasma Samples

The multiplex pre-amplification methods described above were tested onDNA isolated from human plasma samples and treated with bisulfite, asdescribed in Example 1.

Experimental conditions:

-   -   1. Sample source:        -   Extracted and bisulfite-treated 75 plasma samples from            patients with colorectal cancer or stomach cancer, or from            normal patients—2 mL each.    -   2. 50 μL PCR reactions,    -   3. Targets regions tested: NDRG4, BMP3, SFMBT2, VAV3, ZDHHCl,        and 3-actin,    -   4. Reaction conditions used for both pre-amplification and        PCR-flap assay:        -   7.5 mM MgCl2,        -   10 mM MOPS,        -   0.3 mM Tris-HCl, pH 8.0,        -   0.8 mM KCl,        -   0.1 μg/μL BSA,        -   0.0001% Tween-20,        -   0.0001% IGEPAL CA-630,        -   250 μM dNTP)        -   GoTaq polymerase at 0.025 U/μl,        -   Primer pairs for bisulfite-converted NDRG4, BMP3, SFMBT2,            VAV3, ZDHHCl, and β-actin, as shown in FIGS. 5A-5F, at 500            nM each primer.

Pre-amplification cycling was as shown below:

Pre-Amplification Reaction Cycle: Stage Temp/Time #of CyclesPre-incubation 95° C./5′ 1 Amplification 1 95° C./30″ 10 68° C./30″ 72°C./30″ Cooling 40° C./30″ 1

After PCR, 10 μL of the amplification reaction was diluted to 100 μL in10 mM Tris, 0.1 mM EDTA, and 10 μL of the diluted amplification productare used in a standard PCR-flap assay, as described in Example 2.

The data are shown in FIGS. 10A-10I. FIGS. 10A-10C compare the resultsusing the multiplex pre-amplification plus the PCR-flap assay to theresults from the same samples in which no pre-amplification isperformed. FIGS. 10D-10F show the percent methylation calculated foreach sample using the multiplex pre-amplification plus the PCR-flapassay, and FIGS. 10G-10I shows the percent recovery of the input strandsin the multiplex pre-amplification plus the PCR-flap assay, as comparedto the results from the same samples using the PCR-flap assay with nopre-amplification step. Using 3 markers (VAV3, SFMBT2, ZDHHCl) forcolorectal cancer, these data showed 92% sensitivity (23/25), at 100%specificity.

Embodiments of the technology disclosed herein offer at least 100-foldor greater sensitivity for detecting DNA from blood, e.g., 2.5 copiesfrom 4 mL of plasma, compared to 250 copies using the QuARTS PCR flapassay without pre-amplification.

Example 8 An Exemplary Protocol for Complete Blood-to-Result Analysis ofPlasma DNA

An example of a complete process for isolating DNA from a blood samplefor use, e.g., in a detection assay, is provided in this example.Optional bisulfite conversion and detection methods are also described.

I. Blood Processing

Whole blood is collected in anticoagulant EDTA or Streck Cell-Free DNABCT tubes. An exemplary procedure is as follows:

-   -   1. Draw 10 mL whole blood into vacutainers tube (anticoagulant        EDTA or Streck BCT), collecting the full volume to ensure        correct blood to anticoagulant ratio.    -   2. After collection, gently mix the blood by inverting the tube        8 to 10 times to mix blood and anticoagulant and keep at room        temperature until centrifugation, which should happen within 4        hours of the time of blood collection.    -   3. Centrifuge blood samples in a horizontal rotor (swing-out        head) for 10 minutes at 1500 g (±100 g) at room temperature. Do        not use brake to stop centrifuge.    -   4. Carefully aspirate the supernatant (plasma) at room        temperature and pool in a centrifuge tube. Make sure not to        disrupt the cell layer or transfer any cells.    -   5. Carefully transfer 4 mL aliquots of the supernatant into        cryovial tubes.    -   6. Close the caps tightly and place on ice as soon as each        aliquot is made. This process should be completed within 1 hour        of centrifugation.    -   7. Ensure that the cryovials are adequately labeled with the        relevant information, including details of additives present in        the blood.    -   8. Specimens can be kept frozen at −20° C. for a maximum of 48        hours before transferring to a −80° C. freezer.

II. Preparation of a Synthetic Process Control DNA

Complementary strands of methylated zebrafish DNA are synthesized havingthe sequences as shown below using standard DNA synthesis methods suchas phosphoramidite addition, incorporating 5-methyl C bases at thepositions indicated. The synthetic strands are annealed to create adouble-stranded DNA fragment for use as a process control.

A. Annealing and Preparation of Concentrated Zebra Fish (ZF-RASS F1180Mer) Synthetic Process Control

Oligo Name Oligo Sequence Zebrafish 5-TCCAC/iMe-dC/GTGGTGCCCACTCTGGACARASSF1 me GGTGGAGCAGAGGGAAGGTGGTG/iMe-dC/GCA syntheticTGGTGGG/iMe-dC/GAG/iMe-dC/G/iMe-dC/ TargetGTG/iMe-dC/GCCTGGAGGACCC/iMe-dC/GA SenseTTGGCTGA/iMe-dC/GTGTAAACCAGGA/iMe- StranddC/GAGGACATGACTTTCAGCCCTGCAGCCAGAC ACAGCTGAGCTGGTGTGACCTGTGTGGAGAGTTCATCTGG-3 (SEQ ID NO 71) Zebrafish 5-CCAGATGAACTCTCCACACAGGTCACACCAGCRASSF1 me TCAGCTGTGTCTGGCTGCAGGGCTGAAAGTCATG syntheticTCCT/iMe-dC/GTCCTGGTTTACA/iMe-dC/G TargetTCAGCCAAT/iMe-dC/GGGGTCCTCCAGG/ Anti-SenseiMe-dC/GCA/iMe-dC/G/iMe-dC/GCT/ StrandiMe-dC/GCCCACCATG/iMe-dC/GCACCACCT TCCCTCTGCTCCACCTGTCCAGAGTGGGCACCA/iMe-dC/GGTGGA-3 (SEQ ID NO 72)

-   -   1. Reconstitute the lyophilized, single stranded        oligonucleotides in 10 mM Tris, pH 8.0, 0.1 mM EDTA, at a        concentration of 1 μM.    -   2. Make 10× Annealing Buffer of 500 mM NaCl, 200 mM Tris-HCl pH        8.0, and 20 mM MgCl2.    -   3. Anneal the synthetic strands

In a total volume of 100 μL, combine equimolar amounts of each of thesingle-stranded oligonucleotides in 1× annealing buffer, e.g., as shownin the table below:

Final Conc. Volume Stock (copies/μl in 1 ml added Component Conc. finalvolume) (μL) Zebrafish RASSF1 me 1 μM 1.0E+10  16.6 synthetic TargetSense Strand Zebrafish RASSF1 me 1 μM 1.0E+10  16.6 synthetic TargetAnti-Sense Strand Annealing Buffer 10X NA  10.0 Water NA NA  56.8 totalvol. 100.0 μL

-   -   4. Heat the annealing mixture to 98° C. for 11-15 minutes.    -   5. Remove the reaction tube from the heat and spin down briefly        to collect condensation to bottom of tube.    -   6. Incubate the reaction tube at room temp for 10 to 25 minutes.    -   7. Add 0.9 mL fish DNA diluent (20 ng/mL bulk fish DNA in Te (10        mM Tris-HCl pH8.0, 0.1 mM EDTA)) to adjust to the concentration        of zebrafish RASSF1 DNA fragment to 1.0×10¹⁰ copies/μl of        annealed, double-stranded synthetic zebrafish RASSF1 DNA in a        carrier of genomic fish DNA.    -   8. Dilute the process control to a desired concentration with 10        mM Tris, pH 8.0, 0.1 mM EDTA, e.g., as described in the table        below, and store at either −20° C. or −80° C.

Target Total Initial Concentration Addition Te Volume FinalConcentration 1.00E+10 copies/μL 10 μL 990 μL 1000 μL 1.00E+08 copies/μL1.00E+08 copies/μL 10 μL 990 μL 1000 μL 1.00E+06 copies/μL

B. Preparation of 100× Stock Process Control (12,000 Copies/μL ZebraFish RASSF1 DNA in 200 ng/μL bulk Fish DNA)

-   -   1. Thaw reagents    -   2. Vortex and spin down thawed reagents    -   3. Add the following reagents into a 50 mL conical tube

Reagent Initial Concentration Final Concentration Volume to add (mL)Stock carrier fish DNA 10 μg/μL 200 ng/μL 0.40 Zebra fish (ZF-RASS F1180mer) 1.00E+06 copies/μL 1.20E+04 copies/μL 0.24 10 mM Tris, pH 8.0,0.1 mM EDTA NA NA 19.36 Total Volume 20.00

-   -   4. Aliquot into labeled 0.5 mL tubes and store @ −20° C.        C. Preparation of 1× Stock of Process Control (120 Copies/μL        Zebra Fish RASSF1 DNA in 2 ng/μL Fish DNA)    -   1. Thaw reagents    -   2. Vortex and spin down thawed reagents    -   3. Add the following reagents into a 50 mL conical tube:

Reagent 1 mL 5 mL 10 mL 100x Zebra Fish Process Control  10 μL  50 μL 100 μL 10 mM Tris, pH 8.0, 0.1 mM EDTA 990 μL 4950 μL 9900 μL

-   -   4. Aliquot 0.3 mL into labeled 0.5 mL tubes and store @ −20° C.        III. DNA Extraction from Plasma    -   1. Thaw plasma, prepare reagents, label tubes, and clean and        setup biosafety cabinet for extraction    -   2. Add 300 μL Proteinase K (20 mg/mL) to one 50 mL conical tube        for each sample.    -   3. Add 2-4 mL of plasma sample to each 50 mL conical tube (do        not vortex).    -   4. Swirl or pipet to mix and let sit at room temp for 5 min.    -   5. Add 4-6 mL of lysis buffer 1 (LB 1) solution to bring the        volume up to approximately 8 mL.

LB1 Formulation:

-   -   0.1 mL of 120 copies/μL of zebrafish RASSF1 DNA process control,        as described above;    -   0.9-2.9 mL of 10 mM Tris, pH 8.0, 0.1 mM EDTA (e.g., use 2.9 mL        for 2 mL plasma samples)    -   3 mL of 4.3 M guanidine thiocyanate with 10% IGEPAL (from a        stock of 5.3 g of IGEPAL CA-630 combined with 45 mL of 4.8 M        guanidine thiocyanate)    -   6. Invert tubes 3 times.    -   7. Place tubes on bench top shaker (room temperature) at 500 rpm        for 30 minutes at room temperature.    -   8. Add 200 μL of silica binding beads [16 μg of particles/μL]        and mix by swirling.    -   9. Add 7 mL of lysis buffer 2 (LB2) solution and mix by        swirling.

Lb2 Formulation:

-   -   4 mL 4.3 M guanidine thiocyanate mixed with 10% IGEPAL    -   3 mL 100% Isopropanol

(Lysis buffer 2 may be added before, after, or concurrently with thesilica binding beads)

-   -   10. Invert tubes 3 times.    -   11. Place tubes on bench top shaker at 500 rpm for 30 minutes at        room temperature.    -   12. Place tubes on capture aspirator and run program with        magnetic collection of the beads for 10 minutes, then        aspiration. This will collect the beads for 10 minutes then        remove all liquid from the tubes.    -   13. Add 0.9 mL of Wash Solution 1 (3 M guanidine hydrochloride        or guanidine thiocyanate, 56.8% EtOH) to resuspend binding beads        and mix by swirling.    -   14. Place tubes on bench top shaker at 400 rpm for 2 minute at        room temperature.        (All subsequent steps can be done on the STARlet automated        platform.)    -   15. Mix by repeated pipetting then transfer containing beads to        96 deep well plate.    -   16. Place plate on magnetic rack for 10 min.    -   17. Aspirate supernatant to waste.    -   18. Add 1 mL of Wash Solution 2 (80% Ethanol, 10 mM Tris pH        8.0).    -   19. Mix for 3 minutes.    -   20. Place tubes on magnetic rack for 10 min.    -   21. Aspirate supernatant to waste.    -   22. Add 0.5 mL of Wash Solution 2.    -   23. Mix for 3 minutes.    -   24. Place tubes on magnetic rack for 5 min.    -   25. Aspirate supernatant to waste.    -   26. Add 0.25 mL of Wash Solution 2.    -   27. Mix for 3 minutes.    -   28. Place tubes on magnetic rack for 5 min.    -   29. Aspirate supernatant to waste.    -   30. Add 0.25 mL of Wash Solution 2.    -   31. Mix for 3 minutes.    -   32. Place tubes on magnetic rack for 5 min.    -   33. Aspirate supernatant to waste.    -   34. Place plate on heat block at 70° C., 15 minutes, with        shaking.    -   35. Add 125 μL of elution buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM        EDTA).    -   36. Incubate 65° C. for 25 minutes with shaking.    -   37. Place plate on magnet and let the beads collect and cool for        8 minutes.    -   38. Transfer eluate to 96-well plate and store at −80° C. The        recoverable/transferable volume is about 100 μL.

IV. Pre-Bisulfite DNA Quantification

To measure DNA in samples using ACTB gene and to assess zebrafishprocess control recovery, the DNA may be measured prior to furthertreatment. Setup a QuARTS PCR-flap assay using 10 μL of the extractedDNA using the following protocol:

-   -   1. Prepare 10× Oligo Mix containing forward and reverse primers        each at 2 μM, the probe and FRET cassettes at 5 μM and dNTP's at        250 μM each. (See below for primer, probe and FRET sequences)

Concentration Oligo Sequence (5′-3′) (μM) ZF RASSF1 UT CGCATGGTGGGCGAG  2 forward primer (SEQ ID NO: 64) ZF RASSF1 UT ACACGTCAGCCAATCGGG   2reverse primer (SEQ ID NO: 65) ZF RASSF1 UT CCACGGACGGCGCGTGCG   5Probe (Arm 3) TTT/3C6/ (SEQ ID NO: 70) Arm 5 FAM /FAM/TCT/BHQ-1/AGC   5FRET CGGTTTTCCGGCTGAGAC GTCCGTGG/3C6/ (SEQ ID NO: 81) ACTB forwardCCATGAGGCTGGTGTAAA   2 primer 3 G (SEQ ID NO: 102) ACTB ReverseCTACTGTGCACCTACTTA   2 primer 3 ATACAC  (SEQ ID NO: 103) ACTB probeCGCCGAGGGCGGCCTTGG   5 with Arm 1 AG/3C6/  (SEQ ID NO: 104) Arm 1/Q670/TCT/BHQ-2/AG   5 QUASAR670 CCGGTTTTCCGGCTGAGA FRET CCTCGGCG/3C6/(SEQ ID NO: 80) dNTP mix 250

-   -   2. Prepare a master mix as follows:

Volume per Component reaction (μL) Water 15.50 10X oligo Mix 3.00 20XQuARTS Enzyme Mix* 1.50 total volume 20.0 *20X enzyme mix contains 1unit/μL GoTaq Hot start polymerase (Promega), 292 ng/μL Cleavase 2.0flap endonuclease(Hologic).

-   -   3. Pipette 10 μL of each sample into a well of a 96 well plate.    -   4. Add 20 μL of master mix to each well of the plate.    -   5. Seal plate and centrifuge for 1 minutes at 3000 rpm.    -   6. Run plates with following reaction conditions on an ABI7500        or Light Cycler 480 real time thermal cycler

QuARTS Assay Reaction Cycle: Ramp Rate (° C. Number of Signal StageTemp/Time per second) Cycles Acquisition Pre-incubation 95° C./3 min 4.41 No Amplification 1 95° C./2 sec 4.4 5 No 63° C./30 sec 2.2 No 70°C./30 sec 4.4 No Amplification 2 95° C./20 sec 4.4 40 No 53° C./1 min2.2 Yes 70° C./30 sec 4.4 No Cooling 40° C./30 sec 2.2 1 No

V. Bisulfite Conversion and Purification of DNA

-   -   1. Thaw all extracted DNA samples from the DNA extraction from        plasma step and spin down DNA.    -   2. Reagent Preparation:

Component Abbreviation Name Formulation BIS SLN Bisulfite Conversion56.6% Ammonium Bisulfite Solution DES SLN Desulfonation 70% Isopropylalcohol, 0.1N NaOH Solution BND BDS Binding Beads Maxwell RNA Beads (16mg/mL), (Promega Corp.) BND SLN Binding Solution 7M Guanidine HCl CNVWSH Conversion Wash 10 mM Tris-HCl, 80% Ethanol ELU BUF Elution Buffer10 mM Tris, 0.1 mM EDTA, pH 8.0

-   -   3. Add 5 μL of 100 ng/μL BSA DNA Carrier Solution to each well        in a deep well plate (DWP).    -   4. Add 80 μL of each sample into the DWP.    -   5. Add 5 μL of freshly prepared 1.6N NaOH to each well in the        DWP(s).    -   6. Carefully mix by pipetting with pipette set to 30-40 μL to        avoid bubbles.    -   7. Incubate at 42° C. for 20 minutes.    -   8. Add 120 μL of BIS SLN to each well.    -   9. Incubate at 66° C. for 75 minutes while mixing during the        first 3 minutes.    -   10. Add 750 μL of BND SLN    -   11. Pre-mix of silica beads (BND BDS) and add of 50 μL of Silica        beads (BND BDS) to the wells of DWP.    -   12. Mix at 30° C. on heater shaker at 1,200 rpm for 30 minutes.    -   13. Collect the beads on a plate magnet for 5 minutes followed        by aspiration of solutions to waste.    -   14. Add 1 mL of wash buffer (CNV WSH) then move the plate to a        heater shaker and mix at 1,200 rpm for 3 minutes.    -   15. Collect the beads on a plate magnet for 5 minutes followed        by aspiration of solutions to waste.    -   16. Add 0.25 mL of wash buffer (CNV WSH) then move the plate to        the heater shaker and mix at 1,200 rpm for 3 minutes.    -   17. Collect the beads on a plate magnet followed by aspiration        of solutions to waste.    -   18. Add of 0.2 mL of desulfonation buffer (DES SLN) and mix at        1,200 rpm for 7 minutes at 30° C.    -   19. Collect the beads for 2 minutes on the magnet followed by        aspiration of solutions to waste.    -   20. Add of 0.25 mL of wash buffer (CNV WSH) then move the plate        to the heater shaker and mix at 1,200 rpm for 3 minutes.    -   21. Collect the beads for 2 minutes on the magnet followed by        aspiration of solutions to waste.    -   22. Add of 0.25 mL of wash buffer (CNV WSH) then move the plate        to the heater shaker and mix at 1,200 rpm for 3 minutes.    -   23. Collect the beads for 2 minutes on the magnet followed by        aspiration of solutions to waste.    -   24. Allow the plate to dry by moving to heater shaker and        incubating at 70° C. for 15 minutes while mixing at 1,200 rpm.    -   25. Add 80 μL of elution buffer (ELU BFR) across all samples in        DWP.    -   26. Incubated at 65° C. for 25 minutes while mixing at 1,200        rpm.    -   27. Manually Transfer eluate to 96 well plate and store at −80°        C.    -   28. The recoverable/transferable volume is about 65 μL.

VI. QuARTS-X for Methylated DNA Detection and Quantification

A. Multiplex PCR (mPCR) Setup:

-   -   1. Prepare a 10× primer mix containing forward and reverse        primers for each methylated marker of interest to a final        concentration of 750 nM each. Use 10 mM Tris-HCl, pH 8, 0.1 mM        EDTA as diluent, as described in the examples above.    -   2. Prepare 10× multiplex PCR buffer containing 100 mM MOPS, pH        7.5, 75 mM MgCl2, 0.08% Tween 20, 0.08% IGEPAL CA-630, 2.5 mM        dNTPs.    -   3. Prepare multiplex PCR master mix as follows:

Volume per reaction Component (μL) Water 9.62 10X Primer Mix (0.75 μMeach) 7.5 mPCR Buffer 7.5 Hot Start GoTaq (5 units/μl) 0.38 total volume25.0

-   -   4. Thaw DNA and spin plate down.    -   5. Add 25 μL of master mix to a 96 well plate.    -   6. Transfer 50 μL of each sample to each well.    -   7. Seal plate with aluminum foil seal (do not use strip caps)    -   8. Place in heated-lid thermal cycler and proceed to cycle using        the following profile, for about 5 to 20 cycles, preferably        about 10 to 13 cycles:

Number of Stage Temp/Time Cycles Pre-incubation 95° C./5 min 1Amplification 1 95° C./30 sec 12 64° C./60 sec Cooling  4° C./hold 1

-   -   9. After completion of the thermal cycling, perform a 1:10        dilution of amplicon as follows:        -   a. Transfer 180 μL of 10 mM Tris-HCl, pH 8, 0.1 mM EDTA to            each well of a deep well plate.        -   b. Add 20 μL of amplified sample to each pre-filled well.        -   c. Mix the diluted samples by repeated pipetting using fresh            tips and a 200 μL pipettor (be careful not to generate            aerosols).        -   d. Seal the diluted plate with a plastic seal.        -   e. Centrifuge the diluted plate at 1000 rpm for 1 min.        -   f. Seal any remaining multiplex PCR product that has not            been diluted with a new aluminum foil seal. Place at −80° C.

B. QuARTS Assay on Multiplex-Amplified DNA:

-   -   1. Thaw fish DNA diluent (20 ng/μL) and use to dilute plasmid        calibrators (see, e.g., U.S. patent application Ser. No.        15/033,803, which is incorporated herein by reference) needed in        the assay. Use the following table as a dilution guide:

Initial Plasmid Final plasmid μL of μL of total Concentration,Concentration, plasmid diluent to volume, copies per μL copies per μL toadd add μL 1.00E+05 1.00E+04 5 45 50 1.00E+04 1.00E+03 5 45 50 1.00E+031.00E+02 5 45 50 1.00E+02 1.00E+01 5 45 50

-   -   2. Prepare 10× triplex QuARTS oligo mix using the following        table for markers A, B, and C (e.g., markers of interest, plus        run control and internal controls such as β-actin or B3GALT6        (see, e.g., U.S. Pat. Appln. Ser. No. 62/364,082, incorporated        herein by reference).

Concen- tration Oligo Sequence (5′-3′) (μM) Marker A Forward primer NA  2 Marker A Reverse primer NA   2 Marker A probe-Arm 1 NA   5Marker B Forward primer NA   2 Marker B Reverse primer NA   2Marker B probe-Arm 5 NA   5 Marker C Forward primer NA   2Marker C Reverse primer NA   2 Marker C probe-Arm 3 NA   5 A1 HEX FRET/HEX/TCT/BHQ-1/   5 AGCCGGTTTTCCGGC TGAGACCTCGGCG/ 3C6/ (SEQ ID NO: 80)A5 FAM FRET /FAM/TCT/BHQ-1/   5 AGCCGGTTTTCCGGC TGAGACGTCCGTGG/ 3C6/(SEQ ID NO: 81) A3 QUASAR-670 FRET /Q670/TCT/BHQ-2/   5 AGCCGGTTTTCCGGCTGAGACTCCGCGTC/ 3C6/ (SEQ ID NO: 82) dNTP mix 250

For example, the following might be used to detect bisulfite-treatedβ-actin, B3GALT6, and zebrafish RASSF1 markers:

Concen- Oligo tration Description Sequence (5′-3′) (μM) ZF RASSF1 BTTGCGTATGGTGGGCGAG    2 Forward primer (SEQ ID NO: 64) ZF RASSF1 BTCCTAATTTACACGTCAA    2 Reverse primer CCAATCGAA (SEQ ID NO: 68)ZF RASSF1 BT CCACGGACGGCGCGTGC    5 probe-Arm 5 GTTT/3C6/ (SEQ ID NO: 70) B3GALT6 Forward GGTTTATTTTGGTTTTT    2 primerTGAGTTTTCGG  (SEQ ID NO: 73) B3GALT6 Reverse TCCAACCTACTATATTT    2primer ACGCGAA  (SEQ ID NO: 74) B3GALT6 probe- CGCCGAGGGCGGATTTA    5Arm 1 GGG/3C6/  (SEQ ID NO: 76) BTACT Forward GTGTTTGTTTTTTTGAT    2primer TAGGTGTTTAAGA (SEQ ID NO: 77) BTACT Reverse CTTTACACCAACCTCAT   2 primer AACCTTATC  (SEQ ID NO: 78) BTACT probe- GACGCGGAGATAGTGTT   5 Arm 3 GTGG/3C6/  (SEQ ID NO: 79) Arm 1 HEX FRET /HEX/TCT/BHQ-1/   5 AGCCGGTTTTCCGGCTG AGACCTCGGCG/3C6/ (SEQ ID NO: 80) Arm 5 FAM FRET/FAM/TCT/BHQ-1/    5 AGCCGGTTTTCCGGCTG AGACGTCCGTGG/3C6/ (SEQ ID NO: 81)Arm 3 QUASAR- /Q670/TCT/BHQ-2/    5 670 FRET AGCCGGTTTTCCGGCTGAGACTCCGCGTC/3C6/ (SEQ ID NO: 82) dNTP mix 2500

-   -   3. Prepare a QuARTS flap assay master mix using the following        table:

Volume per Component reaction (μL) Water 15.5 10X Triplex Oligo Mix 3.020X QuARTS Enzyme mix 1.5 total volume 20.0 *20X enzyme mix contains 1unit/μL GoTaq Hot start polymerase (Promega), 292 ng/μL Cleavase 2.0flap endonuclease (Hologic).

-   -   4. Using a 96 well ABI plates, pipette 20 μL of QuARTS master        mix into each well.    -   5. Add 10 μL of appropriate calibrators or diluted mPCR samples.    -   6. Seal plate with ABI clear plastic seals.    -   7. Centrifuge the plate using 3000 rpm for 1 minute.    -   8. Place plate in ABI thermal cycler programmed to run the        following thermal protocol then start the instrument

QuARTS Reaction Cycle: Ramp Rate (° C. Number of Signal Stage Temp/Timeper second) Cycles Acquisition Pre-incubation 95° C./3 min 4.4 1 noneAmplification 1 95° C./2 sec 4.4 5 none 63° C./30 sec 2.2 none 70° C./30sec 4.4 none Amplification 2 95° C./20 sec 4.4 40 none 53° C./1 min 2.2Yes 70° C./30 sec 4.4 none Cooling 40° C./30 sec 2.2 1 none

Example 9 Comparison of Chaotropic Salts in First Wash Solution

During development of the technology, the effects of using differentchaotropic salts, e.g., guanidine thiocyanate vs. guanidinehydrochloride in the first wash solution were compared. DNA wasextracted from plasma samples as described in Example 7, with eitherguanidine thiocyanate-ethyl alcohol or guanidine hydrochloride-ethylalcohol used as a first wash solution (i.e., 57% ethyl alcohol witheither 3 M guanidine hydrochloride or 3 M guanidine thiocyanate). Thesamples were otherwise processed as described in Example 7 and a portionof the DNA was bisulfite-converted. The amount of resulting unconvertedDNA was measured by detection of the process control and β-actin (ACTB)using a QuARTS PCR flap assay, as described above, and thebisulfite-converted DNA was measured by detection of the processcontrol, B3GALT6, and β-actin (BTACT) using a multiplexpre-amplification and QuARTS PCR-flap assay, as described above. Theresults are shown in FIGS. 11A-11C (process control data not shown).These data show that both solutions produced acceptable DNA yields, withthe guanidine thiocyanate-ethanol producing higher yields.

Example 10 Comparison of Ethyl Alcohol with Guanidine Thiocyanate orGuanidine Hydrochloride to Ethyl Alcohol with Buffer in a First WashStep

During development of the technology, the effects of using a mixture ofethyl alcohol (ethanol) with a chaotropic salt solution, e.g., guanidinethiocyanate (GTC) or guanidine hydrochloride (GuHCl) in the first washstep of the plasma DNA extraction described in Example 7, part III i.e.,using 57% ethyl alcohol with 3 M guanidine hydrochloride (wash solution1 in Example 7, part III) or 50% ethyl alcohol with 2.4 M guanidinethiocyanate, was compared to using 80% ethyl alcohol with 10 mM TrisHCl, pH 8.0 (wash solution 2 in Example 7, part III) in the first washstep. The 80% ethanol-Tris buffer solution was used in the subsequentwash steps, as described in Example 7.

Eight replicates were performed for each set of wash conditions. Thesamples were otherwise processed as described in Example 7 and the DNAwas not treated with bisulfite. The amount of resulting DNA was measuredby detection of β-actin (ACTB) using a QuARTS PCR flap assay, asdescribed above. The results (mean of DNA strands detected) are shown inthe table below. These data show that use of ethyl alcohol with eitherguanidine thiocyanate or guanidine hydrochloride in the first wash step,followed by additional washes with the ethanol-buffer wash, producedhigher yields than the use of the ethanol-buffer wash for all washsteps.

Wash Condition Mean SD CV Ethanol-Tris buffer 1099 50.80 4.62Ethanol-GuHCl 1434 76.49 5.33 Ethanol-GTC 1416 189.45 13.38

Example 11 Test of Addition of Lysis Reagent in One Step or Two Step

During development of the technology, the effects of adding the lysisreagent at one or two steps in the isolation procedure were compared.Using aliquots of 2 mL or 4 mL from 6 different plasma samples, thefirst procedure comprised adding 7 mL of a lysis reagent of 4.3 Mguanidine thiocyanate with 10% IGEPAL with proteinase K and a processcontrol as described in Example 1, incubation of theplasma/protease/process control mixture at 55° C. for 60 min, followedby addition of isopropanol. The second procedure comprised adding onealiquot of 3 mL of 4.3 M guanidine thiocyanate with 10% IGEPAL with theprotease and process control, and a further aliquot of 4 mL added afterthe 55° C. incubation, along with the addition of isopropanol. Thesamples were then further incubated at 30° C. for 30 min., thenprocessed as described in Example 1. A portion of the resulting DNA wasbisulfite-converted as described.

The amount of resulting unconverted DNA was measured by detection of theprocess control and β-actin (ACTB) using a QuARTS assay, as describedabove, and the bisulfite-converted DNA was measured by detection of theprocess control, B3GALT6, and β-actin (BTACT) using a multiplexpre-amplification and QuARTS PCR-flap assay, as described above. Theresults are shown in FIGS. 12A-12C (process control data not shown). Theaverage fold difference in yield for each tested marker and for theprocess control (PC) is shown below:

Average fold difference of 2 additions vs. 1 addition UnconvertedBisulfite-converted PC ACTB PC B3GALT BTACT 1.07 1.12 1.04 1.12 1.20

These data show that addition of the lysis reagent in two steps, withthe first in the absence of isopropanol and the second added incombination with isopropanol, produces higher yields of detectable DNA.

Example 12 TELQAS Assay Testing

The following experiments were performed in order to test whethermodifying the length and/or stability of the target specific region ofthe probe of the QuARTS assay done at a higher temperature results in animproved performance compared to QuARTS. In this example, the meltingtemperature of the target specific region is calculated to beapproximately 63° C.

In this example, the modified “hotter” QuARTS assay is referred to asthe LQAS assay (for “Long Probe Quantitative Amplified Signal”). TheLQAS probes have a target specific region that have a Tm of about 63° C.The combined pre-amplification and LQAS assay is referred to as theTELQAS assay (for “Target Enrichment Long probe Quantitative AmplifiedSignal”). In the QuARTS assays described below, the flapoligonucleotides have a target specific region of 12 bases. In the LQASassays, the flap oligonucleotides have a target specific region of atleast 13 bases.

For this test, methylated regions within the loci ACP1, SPINT2,CCNJ_3707, CCNJ_3124 and B3GALT6 were selected and the primers andprobes shown below were designed.

AACP1: hg19_dna range=chr2:263991-264161, strand=+ QuARTS Design:(SEQ ID NO: 105)

TTTCGCGGGATAAAAATTACGCGTTCGTCGG (SEQ ID NO: 106) ACP1_FPGCGCGTTGTTTCGTTTCG (SEQ ID NO: 107) ACP1_RP CGTCACCTACCGCAAATACG(SEQ ID NO: 108) ACP1_Pb_A5 CCACGGACG GCGGATAAGGAG/3C6/ (SEQ ID NO: 109)FRET FAM A5  5′d-FAM-TCT-BHQ-1-AGCCGGTTTTCCGGCTGAGACGTCCGTGG-C6 3′LQAS Design: (SEQ ID NO: 110)

TTTCGCGGGATAAAAATTACGCGTTCGTCGG (SEQ ID NO: 111) ACP1_FPGCGCGTTGTTTCGTTTCG (SEQ ID NO: 112) ACP1_RP CGTCACCTACCGCAAATACG(SEQ ID NO: 113) ACP1_Pb_A5_63 AGGCCACGGACG GCGGATAAGGAGGTTTTAGC/3C6/(SEQ ID NO: 114) FRET FAM LQAS A5 5′d-FAM-TCT-BHQ-1-AGCCGGTTTTCCGGCTGAGACGTCCGTGGCCT-C6 3′SPINT2: hg19_dna range=chr19:38755084-38755174, strand=+ QuARTS Design:(SEQ ID NO: 115)

GGTGCGTTTCGTTTTTTGTTT (SEQ ID NO: 116) SPINT2_FP GGGAGCGGTCGCGTAG(SEQ ID NO: 117) SPINT2_RP GCACCTAACTAAACAAAACGAACTAAAC (SEQ ID NO: 118)SPINT2_Pb_A1 CGCCGAGG CGCAAACGCAAA/3C6/ (SEQ ID NO: 119) FRET HEX A1 5′d-HEX-TCT-BHQ-1-AGCCGGTTTTCCGGCTGAGACCTCGGCG-C6 3′ LQAS Design:(SEQ ID NO: 120)

GGTGCGTTTCGTTTTTTGTTT (SEQ ID NO: 121) SPINT2_FP GGGAGCGGTCGCGTAG(SEQ ID NO: 122) SPINT2_RP GCACCTAACTAAACAAAACGAACTAAAC (SEQ ID NO: 123)SPINT2_Pb_A1_63  CGCGCCGAGG CGCAAACGCAAAAAACAAAC/3C6/ (SEQ ID NO: 124)FRET HEX LQAS A1  5′d-HEX-TCT-BHQ-1-AGCCGGTTTTCCGGCTGAGACCTCGGCGCG-C6 3′CCNJ_3707: hg19_dna range=chr10:97803689-97803799 5′pad=0 3′pad=0 strand=+QuARTS Design: (SEQ ID NO: 125)

AGGGCGGGAGAATTTTAGTTTCGGACGTAGGGAGTTTTAGT (SEQ ID NO: 126) CCNJ_3707_FPGCGTTTTTTTTTAGCGGGGTTA (SEQ ID NO: 127) CCNJ_3707_RPCCGAAACTAAAATTCTCCCGC (SEQ ID NO: 169) CCNJ_3707_Pb_A1CGCCGAGG ATGAGCGTGTTA\3C6\ (SEQ ID NO: 128) FRET HEX A1 5′d-HEX-TCT-BHQ-1-AGCCGGTTTTCCGGCTGAGACCTCGGCG-C6 3′ LQAS Design:(SEQ ID NO: 129)

AGGGCGGGAGAATTTTAGTTTCGGACGTAGGGAGTTTTAGT (SEQ ID NO: 130) CCNJ_3707_FPGCGTTTTTTTTTAGCGGGGTTA (SEQ ID NO: 131) CCNJ_3707_RPCCGAAACTAAAATTCTCCCGC (SEQ ID NO: 132) CCNJ_3707_Pb_A1_63CGCGCCGAGG ATGAGCGTGTTATTTTTTTTCGT/3C6/ (SEQ ID NO: 133)FRET HEX LQAS A1 5′d-HEX-TCT-BHQ-1-AGCCGGTTTTCCGGCTGAGACCTCGGCGCG-C6 3′CCNJ_3124: hg19 dna_range=chr10:97803124-97803203 5′pad=0 3′pad=0 strand=-QuARTS Design: (SEQ ID NO: 134)

TTGGTTTGGT (SEQ ID NO: 135) CCNJ_3124_FP CGGTTTTCGTTTGGGTACG(SEQ ID NO: 136) CCNJ_3124_RP CCAACCCAAACCACGCC (SEQ ID NO: 137)CCNJ_3124_Pb_A5 CCACGGACG CGCGCCGTACGA\3C6\ (SEQ ID NO: 138) FRET FAM A55′d-FAM-TCT-BHQ-1-AGCCGGTTTTCCGGCTGAGACGTCCGTGG-C6 3′ LQAS Design:(SEQ ID NO: 139)

TTGGTTTGGT (SEQ ID NO: 140) CCNJ_3124_FP CGGTTTTCGTTTGGGTACG(SEQ ID NO: 141) CCNJ_3124_RP CCAACCCAAACCACGCC (SEQ ID NO: 142)CCNJ_3124_Pb_A5_63 AGGCCACGGACG CGCGCCGTACGAAAT/3C6/  (SEQ ID NO: 143)FRET FAM LQAS A5 5′d-FAM-TCT-BHQ-1-AGCCGGTTTTCCGGCTGAGACGTCCGTGGCCT-C6 3′B3GALT6: hg19_dna_range=chr1: 1163595-1163733 strand=+ QuARTS Design:(SEQ ID NO: 144)

(SEQ ID NO: 145) B3GALT6_FP_V3 AGGTTTATTTTGGTTTTTTGAGTTTTCG(SEQ ID NO: 146) B3GALT6_RP TCCAACCTACTATATTTACGCGAA (SEQ ID NO: 147)B3GALT6_Pb_A3_v2 GACGCGGAG GGCGGATTTAGG/3C6/ (SEQ ID NO: 148)FRET Q670 A3 5′d-Q670-TCT-BHQ-2-AGCCGGTTTTCCGGCTGAGACTCCGCGTC-C6 3′LQAS Design: (SEQ ID NO: 149)

(SEQ ID NO: 145) B3GALT6_FP_V3 AGGTTTATTTTGGTTTTTTGAGTTTTCG(SEQ ID NO: 151) B3GALT6_RP TCCAACCTACTATATTTACGCGAA (SEQ ID NO: 152)B3GALT6_Pb_A3_63 ACGGACGCGGAG GCGGATTTAGGGTATTTAAGGAG/3C6/ (SEQ ID NO: 153) FRET Q670 LQAS A3 5′d-Q670-TCT-BHQ-2-AGCCGGTTTTCCGGCTGAGACTCCGCGTCCGT-C6

Experiment 1

This experiment was designed to test dilutions of individual pUC57plasmids containing inserts of ACP1, SPINT2 and CCNJ (diluted to 500,250, and 100 strands per 50 l) in both the QuARTs and TELQAS assays.This was done to evaluate the performance of the TELQAS assay incomparison to the QuARTs assay. In these assays, dilutions of theindividual pUC57 plasmids (containing inserts of ACP1, SPINT2 and CCNJ)were made and amplified 12 cycles with primer mixes. Afteramplification, the PCR product was diluted 10× and LQAS and QuARTSassays were setup, as shown below.

The reactions were set up as follows:

Component μL/Rxn Molecular Biology Grade (MBG) 15.5 H₂O 10X Oligo Mix3.00 20X Enzyme Mix + 2XCL 1.5 Total Vol. Master Mix (μL) 20.0 Sample(μL) 10 Final Rxn Vol (μL): 30

The QuARTs reactions were subjected to the following thermocyclingconditions:

Ramp Temp/ Rate (° C. QuARTS Reaction Cycle: Time second −1) AcquisitionPre-incubation 95° C./3′ 4.4 none Amplification 95° C./20″ 4.4 none 63°C./30″ 2.2 none 70° C./30″ 4.4 none Amplification 95° C./20″ 4.4 none53° C./1′ 2.2 single 70° C./30″ 4.4 none Cooling 40° C./30″ 2.2 none

The TELQAS reactions were subjected to the following thermocyclingconditions:

TELQAS Cycling Ramp Rate Stage Temp/Time (° C./sec) # Cycles AcquisitionDenaturation 95° C./3′ 4.4 1 None Amplification 95° C./20″ 4.4 40 None63° C./1′ 2.2 Single 70° C./30″ 4.4 None Cooling 40° C./30″ 2.2 1 None

Representative results obtained from these tests are shown in FIG. 14.The left hand figures show the signals produced in these assays plottedagainst the number of cycles. The right hand columns show standardcurves. This data shows that:

For all tests:

-   -   TELQAS and QuARTS result in linear standard curves;    -   TELQAS assays result in faster reactions and lower Cps than        QuARTS assays;    -   Neither TELQAS nor QuARTS generate a non-specific signal.

Experiment 2

This experiment was designed to determine the sensitivity of the QuARTSand TELQAS assays for detecting liver-related methylation markers on 285age-matched plasma samples from normal individuals and patients withhepatocellular carcinoma (HCC) and cirrhosis. The strands level of eachmarker was compared to the reference gene, B3GALT6. The performance ofthe TELQAS assay was compared to the performance of the QuARTs assay bycomparing strands per reaction for each sample tested.

In these assays, the target loci were amplified 12 cycles with primermixes. After amplification, the PCR product was diluted 10× and LQAS andQuARTS assays were setup, as shown below.

Component μL/Rxn MBG H₂O 15.5 10X Oligo Mix 3.00 20X Enzyme Mix + 2XCL1.5 Total Vol. Master Mix (μL) 20.0 Sample (μL) 10 Final Rxn Vol (μL):30

The QuARTs reactions were subjected to the following thermocyclingconditions:

Ramp Rate Temp/ (° C. QuARTS Reaction Cycle: Time second −1) AcquisitionPre-incubation 95° C./3′ 4.4 none Amplification 95° C./20″ 4.4 none 63°C./30″ 2.2 none 70° C./30″ 4.4 none Amplification 95° C./20″ 4.4 none53° C./1′ 2.2 single 70° C./30″ 4.4 none Cooling 40° C./30″ 2.2 none

The TELQAS reactions were subjected to the following thermocyclingconditions:

TELQAS Cycling Ramp Rate Stage Temp/Time (° C./sec) # Cycles AcquisitionDenaturation 95° C./3′ 4.4 1 None Amplification 95° C./20″ 4.4 40 None63° C./1′ 2.2 Single 70° C./30″ 4.4 None Cooling 40° C./30″ 2.2 1 None

Representative results obtained from these tests are shown in FIG. 15.This data shows that there is good correlation between the QuARTS andTELQAS platforms with a marginal improvement in resultingstrands/reaction with the TELQAS assay.

Example 13 Use of Probes Having an Allele-Specific Region that ContainsInosine

Incorporating deoxyinosine, or another base that is capable of makingnon-Watson-Crick base pairs, into the target specific region of a probecould, in theory, provide a longer probe without increasing its Tm, inan experimental approach referred to as “Z-QuARTS.” Such probes couldbeassayed using the conditions used for QuARTs, particularly with regardto the annealing temperature in the PCR cycle (see above). The followingexperiments were performed in order to examine the effect, if any, oflengthening the target specific region and substituting one or more ofthe nucleotides of the target specific region with a deoxyinosine. Inthe QuARTS assays described below, the flap oligonucleotides have atarget specific region of 12 bases. In the Z-QuARTs assays, the flapoligonucleotides have a target specific region of at least 13 bases.

Experiment 1

The following designs were tested for HOXB2 (hg19_dnarange=chr17:46620545-46620639 strand=-):

QuARTS Design: (SEQ ID NO: 154)

TTTTGTTTTTAGTTATT (SEQ ID NO: 155) HOXB2_FP GTTAGAAGACGTTTTTTCGGGG (SEQID NO: 156) HOXB2_RP AAAACAAAAATCGACCGCGA (SEQ ID NO: 157) HOXB2_Pb_A1Probe ASR = 12. CGCCGAGG GCGTTAGGATTT/3C6/ (SEQ ID NO: 158) FRET HEX A15′d-HEX-TCT-BHQ-1-AGCCGGTTTTCCGGCTGAGACCTCGGCG-C6 3′ Z-QuARTS Design:(SEQ ID NO: 154)

TTTTGTTTTTAGTTATT (SEQ ID NO: 155) HOXB2_FP GTTAGAAGACGTTTTTTCGGGG ( SEQID NO: 156) HOXB2_RP AAAACAAAAATCGACCGCGA (SEQ ID NO: 159)HOXB2_Pb_A1_dl_16 Probe ASR = 16.

(dl=deoxy inosine) (Note, di base pairs to Adenine in this design) (SEQID NO: 158) FRET HEX A15′d-HEX-TCT-BHQ-1-AGCCGGTTTTCCGGCTGAGACCTCGGCG-C6 3′

In this experiment, a serial dilution of a pUC57 plasmid containinginsert of HOXB2 was made and tested using oligo mixes made for bothHOXB2.

Both reactions were set up in the following way:

μL for Master Mix Component μL/Rxn 16 rxns MBG H₂O 15.5 260.4 10X OligoMix 3.00 50.4 20X Enzyme Mix 1.5 25.2 Total Vol. Master Mix (μL) 20.0336.0 Sample (μL) 10 Final QuARTs Rxn Vol (μL): 30 30

Both reactions were subjected to the following thermocycling conditions:

QuARTs Cycling Ramp Rate Stage Temp/Time (° C./sec) # Cycles AcquisitionDenaturation 95° C./3′ 4.4 1 None Amplification 1 95° C./20″ 4.4 5 None63° C./30″ 2.2 None 70° C./30″ 4.4 None Amplification 95° C./20″ 4.4 40None 53° C./1′ 2.2 Single 70° C./30″ 4.4 None Cooling 40° C./30″ 2.2 1None

Representative results obtained from these tests are shown in FIG. 16.This data shows that the Z-QuARTS assay results in a linear standardcurve (R²=0.9934). The Z-QuARTS assay results in approximately 2 cyclesfaster performance. The amplification curves of Z-QuARTS showed nobackground signal in the no target control and showed faster signalgeneration compared to QuARTS.

Experiment 2

The purpose of this experiment is to test the Z-QuARTS assay against theconventional QuARTS assay using plasma samples spiked with H520 CCM DNAand pre-amplified.

The following primers and probes were used:

QuARTS-Z Design:HOXI32_BST >hg19_dna range=chr17:46620545-46620639 strand=-(SEQ ID NO: 154)

TTTTGTTTTTAGTTATT (SEQ ID NO: 155) HOXB2_FP  GTTAGAAGACGTTTTTTCGGGG(SEQ ID NO: 156) HOXB2_RP  AAAACAAAAATCGACCGCGA (SEQ ID NO: 159)HOXB2_Pb_A1_dl_16  CGCCGAGG GCGTTAGGATTTA/dl/TT/3C6/ (l=deoxy inosine) (SEQ ID NO: 158) FRET HEX A15′d-HEX-TCT-BHQ-1-AGCCGGTTTTCCGGCTGAGACCTCGGCG-C6 3′ QuARTS Designs:HOXB2_BST >hg19_dna range=chr17:46620545-46620639 strand=-(SEQ ID NO: 154)

TTTTGTTTTTAGTTATT (SEQ ID NO: 155) HOXB2_FP  GTTAGAAGACGTTTTTTCGGGG(SEQ ID NO: 156) HOXB2_RP  AAAACAAAAATCGACCGCGA (SEQ ID NO: 157)HOXB2_Pb_A1  CGCCGAGG GCGTTAGGATTT/3C6/  (SEQ ID NO: 158) FRET HEX A15′d-HEX-TCT-BHQ-1-AGCCGGTTTTCCGGCTGAGACCTCGGCG-C6 3′BARX1_BST >hg19_dna range=chr9:96721498-96721597 strand=- BST:(SEQ ID NO: 160)

GTTGTTCGGACGATCGTATTCGGAG (SEQ ID NO: 161) BARX1_FP CGTTAATTTGTTAGATAGAGGGCG (SEQ ID NO: 162) BARX1_RP_universal TCCGAACAACCGCCTAC (SEQ ID NO: 163) BARX1_Pb_A5_universal CCACGGACG CGAAAAATCCCA/3C6/ (SEQ ID NO: 164) FRET FAM A55′d-FAM-TCT-BHQ-1-AGCCGGTTTTCCGGCTGAGACGTCCGTGG-C6 3′B3GALT6_RG BST: >hg19_dna range=chr1:1163595-1163733 strand=+(SEQ ID NO: 144)

GTAGGTTTTCGCGTAAATATAGTAGGTTGGAAGTGGCGTTTATTATCGGTACGTTTTTTTAG(SEQ ID NO: 165) B3GALT6_FP_V3  AGGTTTATTTTGGTTTTTTGAGTTTTCG(SEQ ID NO: 166) B3GALT6_RP  TCCAACCTACTATATTTACGCGAA (SEQ ID NO: 167)B3GALT6_Pb_A3_V2 GACGCGGAG GGCGGATTTAGG/3C6/ (SEQ ID NO: 166)FRET Q670 A3 5′d-Q670-TCT-BHQ-2-AGCCGGTTTTCCGGCTGAGACTCCGCGTC-C6 3′

In summary, a serial dilution of pUC57 plasmid containing inserts ofHOXB2, BARX1 and B3GALT6 was made and tested alongside a serial dilutionof normal plasma samples spiked with H520 CCM DNA. These samples werepre-amplified and tested using oligo mixes made for both designs, QuARTSand Z-QuARTS containing the HOXB2 probe with deoxy inosine.

Both reactions were set up in the following way:

Master Mix Component μL/Rxn MBG H₂O 15.5 10X Oligo Mix 3.00 20X EnzymeMix 1.5 Total Vol. Master Mix (μL) 20.0 Sample (μL) 10 Final QuARTS RxnVol (μL): 30

Both reactions were subjected to the following thermocycling conditions:

Ramp Rate (° C. QuARTS Reaction Cycle: Temp/Time second −1) AcquisitionPre-incubation 95° C./3′ 4.4 none Amplification 95° C./20″ 4.4 none 63°C./30″ 2.2 none 70° C./30″ 4.4 none Amplification 95° C./20″ 4.4 none53° C./1′ 2.2 single 70° C./30″ 4.4 none Cooling 40° C./30″ 2.2 none

Representative results obtained from these tests are shown in FIG. 17.The calibrator curves and Cps demonstrate that the HOXB2—Z-QuARTS designresults in faster amplification than QuARTS by 2 cycles. In addition,Z-QuARTS was able to detect more HOXB2 strands than QuARTS at each levelof dilution. Z-QuARTS in triplex resulted in a linear standard curve andno background signal was generated in controls that contain no targetDNA.

All publications and patents mentioned in the above specification areherein incorporated by reference in their entireties for all purposes.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which the various embodiments described herein belongs.When definitions of terms in incorporated references appear to differfrom the definitions provided in the present teachings, the definitionprovided in the present teachings shall control.

Various modifications and variations of the described compositions,methods, and uses of the technology will be apparent to those skilled inthe art without departing from the scope and spirit of the technology asdescribed. Although the technology has been described in connection withspecific exemplary embodiments, it should be understood that thetechnology as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the technology that are obvious to those skilled inpharmacology, biochemistry, medical science, or related fields areintended to be within the scope of the following claims.

We claim:
 1. A method of analyzing a sample for multiple target nucleicacids, comprising: a) providing a sample having volume x, the samplecomprising bisulfite-treated DNA suspected of containing one or more ofa plurality of n different target regions, wherein n is at least 3, andwherein at least one of said target regions is a low-copy target that,if present in said sample, is present in said sample at a copy numbersuch that: i) among n fractions of said sample each having a volume ofx/n, said low copy target is absent from one or more of said nfractions, or ii) among n fractions of said sample each having a volumeof x/n, said low copy target in one or more of said n fractions is belowa level of sensitivity of a detection assay for said low copy target; b)treating said volume x of said sample to an amplification reaction underconditions wherein said n different target regions, if present in saidsample, are amplified to form a pre-amplified mixture having volume y;c) partitioning said pre-amplified mixture into a plurality of differentdetection assay reaction mixtures, wherein each detection assay reactionmixture comprises a portion of said pre-amplified mixture that has avolume of y/n or less, and wherein said low-copy target, if present insaid sample at step a), is present in each of said detection assayreaction mixtures; d) conducting a plurality of detection assays withsaid detection assay reaction mixtures, wherein said different targetregions, if present in said sample at step a), are detected in saiddetection assay reaction mixtures, wherein the detection assays arePCR-flap assays that employ flap oligonucleotides that have atarget-specific region of at least 13 bases in length.
 2. The method ofclaim 1, wherein one or more of the flap oligonucleotides used in (d)has a target-specific region comprising one or more nucleotides that arecapable of making non-Watson-Crick base pairs.
 3. The method of claim 1,wherein one or more of the flap oligonucleotides used in (d) has atarget-specific region having a length in the range of 13 to 30 bases.4. The method of claim 1, wherein the one or more of the flapoligonucleotides used in (d) has a target-specific region that has aT_(m) in the range of 60° C. to 70° C. and the detection assay comprisesa denaturation step at least 90° C., an annealing step at a temperaturethat is in the range of 60° C. to 70° C., and an extension step at atemperature in the range of 65° C. to 75° C.
 5. The method of claim 1,wherein said bisulfite treated DNA is from a human subject.
 6. Themethod of claim 5, wherein said sample is prepared from a body fluid. 7.The method of claim 5, wherein said body fluid comprises plasma.
 8. Themethod of claim 7, wherein the sample is prepared from cell-free DNAisolated from plasma.
 9. The method of claim 8, wherein said cell-freeDNA is less than 200 base pairs in length.
 10. The method of claim 8,wherein said cell-free DNA is isolated from said plasma by a methodcomprising: a) combining the plasma sample with: i) protease; and ii) afirst lysis reagent, said first lysis reagent comprising guanidinethiocyanate; and non-ionic detergent; to form a mixture wherein proteinsare digested by said protease; b) to the mixture of step a) adding iii)silica particles, and iv) a second lysis reagent, said second lysisreagent comprising: guanidine thiocyanate; non-ionic detergent; andisopropyl alcohol; under conditions wherein DNA is bound to said silicaparticles; c) separating silica particles with bound DNA from themixture of b); d) to the separated silica particles with bound DNAadding a first wash solution, said first wash solution comprisingguanidine hydrochloride or guanidine thiocyanate and ethyl alcohol; e)separating the silica particles with bound DNA from said first washsolution; f) to the separated silica particles with bound DNA adding asecond wash solution, said second wash solution comprising a buffer andethyl alcohol; g) separating washed silica particles with bound DNA fromsaid second wash solution; and h) eluting DNA from the washed silicaparticles with bound DNA.
 11. The method of claim 10, wherein saidprotease is Proteinase K protease.
 12. The method of claim 6, whereinthe sample is prepared from at least one mL, and/or wherein the volume xof said sample is at least 10 μl, and wherein the volume of said samplein the amplification reaction of step b is at least 20 to 50% of thetotal volume of the amplification reaction.
 13. The method of claim 1,wherein n is at least
 4. 14. A method for analyzing multiple targetnucleic acids in a sample using a PCR pre-amplification and a PCR-flapassay, the method comprising: a) providing bisulfite-treated DNAcomprising a plurality of different target regions in a first reactionmixture comprising PCR amplification reagents, wherein said PCRamplification reagents comprise: i) a plurality of different primerpairs for amplifying said plurality of different target regions, ifpresent in said sample, from said bisulfite-treated DNA; ii)thermostable DNA polymerase; iii) dNTPs; and iv) a buffer comprisingMg⁺⁺ b) exposing said first reaction mixture to thermal cyclingconditions wherein a plurality of different target regions, if presentin the sample, are amplified to produce a pre-amplified mixture, andwherein said thermal cycling conditions are limited to fewer than 20thermal cycles; c) partitioning said pre-amplified mixture into aplurality of PCR-flap assay reaction mixtures, wherein each PCR-flapassay reaction mixture comprises: i) an additional amount of a primerpair selected from said plurality of different primer pairs of step a)i); ii) thermostable DNA polymerase; iii) dNTPs; iv) said buffercomprising Mg⁺⁺ v) a flap endonuclease; vi) a flap oligonucleotide thathas a target-specific region of at least 13 bases in length, and vi) ahairpin oligonucleotide comprising a region that is complimentary to aportion of said flap oligonucleotide; and d) detecting amplification ofone or more different target regions from said bisulfite-treated DNAduring PCR-flap assay reactions.
 15. The method of claim 14, whereinsaid pre-amplified mixture is diluted with a diluent prior to saidpartitioning.
 16. The method of claim 15, wherein the primers in theadditional amount of a primer pair added to the PCR-flap assay reactionmixture are added to a concentration such that the concentration of theadded primers in the PCR-flap assay is essentially the same as theconcentration of the primers of that primer pair in said PCRamplification reagents.
 17. The method of claim 14, wherein said hairpinoligonucleotide comprises a fluorophore moiety.
 18. The method of claim14, wherein said first reaction mixture and/or said plurality ofPCR-flap assay reaction mixtures comprise bulk fish DNA.
 19. The methodof claim 14, wherein said flap endonuclease is a FEN-1 endonuclease. 20.The method of claim 14, wherein said buffer comprising Mg⁺⁺ comprisesbetween about 6 to 10 mM Mg⁺⁺.